Early clinical studies showed that high-dose vitamin C, given by
intravenous and oral routes, may improve symptoms and prolong life
in patients with terminal cancer. Double-blind placebo-controlled
studies of oral vitamin C therapy showed no benefit. Recent
evidence shows that oral administration of the maximum tolerated
dose of vitamin C (18 g/d) produces peak plasma concentrations of
only 220 μmol/L, whereas intravenous administration of the same
dose produces plasma concentrations about 25-fold higher. Larger
doses (50–100 g) given intravenously may result in plasma
concentrations of about 14 000 μmol/L. At concentrations above
1000 μmol/L, vitamin C is toxic to some cancer cells but not to
normal cells in vitro. We found 3 well-documented cases of
advanced cancers, confirmed by histopathologic review, where
patients had unexpectedly long survival times after receiving
high-dose intravenous vitamin C therapy. We examined clinical
details of each case in accordance with National Cancer Institute
(NCI) Best Case Series guidelines. Tumour pathology was verified
by pathologists at the NCI who were unaware of diagnosis or
treatment. In light of recent clinical pharmacokinetic findings
and in vitro evidence of anti-tumour mechanisms, these case
reports indicate that the role of high-dose intravenous vitamin C
therapy in cancer treatment should be reassessed.

Thirty years ago Cameron, Campbell and Pauling reported beneficial
effects of high-dose vitamin C (ascorbic acid) therapy for
patients with terminal cancer.1–4 Subsequent double-blind,
randomized clinical trials at the Mayo Clinic failed to show any
benefit,5,6 and the role of vitamin C in cancer treatment was
discarded by mainstream oncologists.7,8 Vitamin C continues,
however, to be used as an alternative cancer therapy.9,10

A key distinction between conventional, science-based medicine and
alternative therapy is the presence or absence of scientific
plausibility.11 In conventional medicine, the efficacy of
treatment is proven by properly conducted clinical trials. Many
treatments are still used if there is moderately good, albeit
inconclusive evidence of efficacy (“clinical plausibility”),
especially when treatment rationale agrees with biologic facts
(conferring “biological plausibility”).11 Vitamin C is an
alternative cancer therapy because the results obtained in
original studies that suggested clinical benefit were not
confirmed by controlled clinical trials, and the notion that
high-dose vitamin C was selectively toxic to cancer cells was
biologically implausible.

New information is available pertaining to biological
plausibility. Although similar doses of vitamin C were used in the
Cameron–Pauling and Mayo Clinic studies, the Cameron–Pauling
studies combined intravenous and oral administration whereas the
Mayo Clinic studies used only oral administration.1,2,12–14 Recent
pharmacokinetics modeling15 indicates that with oral
administration, even very large and frequent doses of vitamin C
will increase plasma concentrations only modestly, from 70 μmol/L
to a maximum of 220 μmol/L, whereas intravenous administration
raises plasma concentrations as high as 14 000 μmol/L.
Concentrations of 1000–5000 μmol/L are selectively cytotoxic to
tumour cells in vitro,16–20 and emerging evidence indicates that
ascorbic acid at concentrations achieved only by the intravenous
route may function as a pro-drug for hydrogen peroxide delivery to
tissues.20 The in vitro biologic evidence and clinical
pharmacokinetics data confer biological plausibility to the notion
that vitamin C could affect cancer biology and may explain in part
the negative results of the Mayo Clinic trials.13,15,21,22 Thus,
sufficient evidence has accumulated, not to use vitamin C as
cancer treatment, but to further explore the therapeutic concept.
One way to increase the clinical plausibility of alternative
cancer therapies is rigorous, well-documented case reporting, as
laid out in the US National Cancer Institute (NCI) Best Case
Series guidelines
(http://www3.cancer.gov/occam/bestcase.html).23,24 Such case
series might identify alternative therapies that merit further
investigation.23,24

Case reports of apparent responses by malignant disease to
intravenous vitamin C therapy have appeared,25–30 including those
of 2 of the 3 patients presented below.25,26 However, they were
reported without sufficient detail or with incomplete follow-up
for evaluation and without conforming to NCI Best Case Series
guidelines. They also lacked objective pathologic confirmation,
which is a pillar of NCI guidelines. In this article, we use NCI
Best Case Series guidelines to report 3 cases of patients with
usually progressive malignant disease who received intravenous
vitamin C therapy as their only significant cancer therapy and
whose clinical courses were unusually favourable. Original
diagnostic material obtained before treatment with vitamin C was
reviewed by pathologists at the National Institutes of Health
(NIH) who were unaware of the diagnoses and treatments.

Patient 1

This case was previously reported but without long-term follow-up,
without detail, and with no independent pathologic
confirmation.25,26 A 51-year-old woman was found in August 1995 to
have a tumour involving her left kidney. At nephrectomy in
September 1995 this was shown to be a renal cell carcinoma 9 cm in
diameter with thrombus extending into the renal vein. Chest
radiography results were normal, and there was no evidence of
metastatic disease on CT scan of the chest and abdomen. In March
1996 a CT scan of the chest indicated several new, small, rounded
and well-defined soft tissue masses no larger than 5 mm in
diameter; they were judged consistent with metastatic cancer. By
November 1996 chest radiography revealed multiple cannonball
lesions (Fig. 1).

Fig. 1: Chest radiography, November 1996, about 1 month after
intravenous vitamin C therapy was started. Cannonball lesions are
evident in both lung fields, as indicated by the arrows and lines.

The patient declined conventional cancer treatment and instead
chose to receive high-dose vitamin C administered intravenously at
a dosage of 65 g twice per week starting in October 1996 and
continuing for 10 months. She also used other alternative
therapies: thymus protein extract, N-acetylcysteine, niacinamide
and whole thyroid extract (Table 1). In June 1997 chest
radiography results were normal except for one remaining
abnormality in the left lung field, possibly a pulmonary scar
(Fig. 2).

In October 2001 a new mass 3.5 cm in diameter in the anterior
right lung was detected on radiography. A transthoracic biopsy
revealed small-cell carcinoma of the lung. The patient opted for
intravenous vitamin C injections. The lung mass remained constant
in size in radiograpy taken in May and August 2002 but had
increased to 4 cm in views taken in October 2002. In early
November hyponatremia developed. Two weeks later the patient was
admitted to hospital with abdominal distension and constipation.
Barium studies revealed slow transit but no intestinal
obstruction. Results of a CT scan of the abdomen were normal. She
died shortly afterward, and no autopsy was performed.
Histopathologic review of the primary renal tumour at the NIH
confirmed the diagnosis of clear-cell renal carcinoma, type,
nuclear grade III/IV, with the largest diameter measuring 6.5 cm.
The tumour involved the renal vein and hilar perinephric fat.
Pathologic review of the lung tumour biopsy specimen of October
2001 was not conducted at the NIH. Local pathologists diagnosed
this specimen as indicating small-cell lung cancer and not
recurrent metastatic renal cell carcinoma.

This case describes the regression of pulmonary metastatic renal
cancer in a patient receiving high-dose intravenous vitamin C
therapy. According to the NCI Best Case Series guidelines, the
credibility of this case would be increased by biopsy proof that
the multiple slowly growing bilateral cannonball lung nodules in
this patient with known renal cell carcinoma were actually
malignant. However, in this case, the clinical characteristics and
evolution of the pulmonary lesions, in the absence of bacterial
infection or other systemic disease, make any other diagnosis
unlikely. The clinician attending the patient deemed a
confirmatory biopsy to be unnecessary and inappropriate in this
setting. A plausible alternative explanation to the conclusion
that this patient's metastatic renal cell cancer responded to
intravenous vitamin C therapy is that the tumours spontaneously
regressed. Spontaneous regression has been reported in renal cell
cancer, but it is rare, occurring in fewer than 1% of cases and
typically after nephrectomy, radiation to the primary tumour or
primary tumour embolization.31,32 Here, metastatic disease
appeared several months after nephrectomy, rather than regressing
in response to it. As well, the primary cancer was nuclear grade
III/IV and involved the renal vein, factors associated with a
highly unfavourable prognosis.31

Of note, more than 4 years after stopping intravenous vitamin C
therapy and with the renal cell cancer in complete remission,
primary small-cell lung cancer was diagnosed in this patient, who
was a long-standing cigarette smoker. The second cancer did not
respond to high-dose vitamin C therapy. From the clinical history
it appears the tumour remained a constant size for many months and
likely slowly progressed until her death about a year after
diagnosis despite the resumption of intravenous vitamin C therapy.

Patient 2

A 49-year-old man presented to his physician in 1996 with
hematuria and was found at cystoscopy to have a primary bladder
tumour with multiple satellite tumours extending 2–3 cm around it.
Transurethral resection of the primary tumour and its surrounding
tumour satellites was carried out until apparently normal muscle
was reached and the tumour base was fulgurated. The patient
declined systemic or intravesical chemotherapy or radiotherapy and
instead chose intravenous vitamin C treatment. He received 30 g of
vitamin C twice per week for 3 months, followed by 30 g once every
1–2 months for 4 years, interspersed with periods of 1–2 months
during which he had more frequent infusions. Histopathologic
review at the NIH revealed a grade 3/3 papillary transitional cell
carcinoma invading the muscularis propria. Now, 9 years after
diagnosis, the patient is in good health with no symptoms of
recurrence or metastasis. The patient used the following
supplements: botanical extract, chondroitin sulfate, chromium
picolinate, flax oil, glucosamine sulfate, α-lipoic acid,
Lactobacillus acidophilus and L. rhamnosus and selenium (Table 2).

Table 2

Complete or partial bladder removal is the standard treatment for
stage T2 (muscle invasive) bladder cancer, since the presence of
muscle invasion appears to be the best predictor of aggressive
behaviour. When treated only locally, as in this case, invasive
transitional cell bladder cancer almost invariably develops into
clinically apparent local or metastatic disease within a short
period.33–35 There are reports of transurethral tumour resection
being offered as the sole initial therapy in carefully selected
patients with T2 disease. In one report 20% of patients with
muscle invasive bladder cancer treated only with transurethral
resection remained free of recurrent disease after 3–7 years of
follow-up.36 However, such minimal therapy is considered an option
only when the cancer is solitary, well defined and completely
excised as documented by pathologic evaluation,37 whereas this
patient presented with multiple tumours and associated muscle
invasion.

Patient 3

This case was previously reported but without detail and without
independent pathologic confirmation.26,30 A 66-year-old woman was
found in January 1995 to have a large left paraspinal mass medial
to the iliopsoas muscle at the L4–5 level. On imaging the mass
measured 3.5–7 cm transversely and 11 cm in the craniocaudal
direction, with evidence of extension into the posterior
paraspinal muscle and bone invasion. Chest radiography results
were normal. An open biopsy specimen was diagnostic of a diffuse
large B-cell lymphoma. The patient's oncologist recommended local
radiation therapy and chemotherapy. Although she agreed to a
5-week course of local radiation therapy, the patient refused
chemotherapy, electing instead to receive vitamin C intravenously.
She received 15 g of vitamin C twice per week for about 2 months,
15 g once to twice per week for about 7 months, and then 15 g once
every 2–3 months for about 1 year. This began in mid-January 1995
concurrently with the radiation therapy, which was given as AP/PA
parallel opposed 18 MEV x-rays and between 1–18 and 2–28–95, 5040
Centi Gray in 28 fractions delivered to the mid-plane of the body
with 3:2 loading from the back. At this time a left axillary lymph
node 1 cm in diameter and a right axillary lymph node 1.5 cm in
diameter were palpable.

Two weeks later, in early February 1995, the right and left
axillary lymph nodes remained palpable and a new left cervical
lymph node 1 cm in diameter and a new left supraclavicular lymph
node larger than 1 cm were apparent on physical examination.
Intravenous vitamin C therapy continued. Three weeks later the
supraclavicular and cervical lymph nodes were no longer palpable,
the left axillary node had disappeared, and the right axillary
node had decreased in size to less than 1 cm. After a further 3
weeks, in mid-March 1995, there was no lymphadenopathy in the neck
and no palpable axillary lymphadenopathy. In late April 1995 a new
left cervical lymph node was detected, and histopathologic review
identified a biopsy specimen as identical to the original tumour.
The patient once again refused chemotherapy and continued her
program of intravenous vitamin C injections. Two months later, in
June 1995, there was marked left supraclavicular lymphadenopthy 3
cm in size, with shotty right axillary nodes but no adenopathy in
the left axilla. Four months later, in October 1995, a single
right submandibular node was palpable, but the supraclavicular and
all other areas, including the axillas, had no palpable lymph
nodes. In May 1996 a left anterior cervical node 1.5 cm in size
was present, but there was no other adenopathy. Intravenous
vitamin C therapy continued through late December 1996, at which
time the patient was in normal health and had no clinical sign of
lymphoma. The patient remains in normal health 10 years after the
diagnosis of diffuse large B-cell lymphoma, never having received
chemotherapy. The patient used additional products: β-carotene,
bioflavonoids, chondroitin sulfate, coenzyme Q10,
dehydroepiandrosterone, a multiple vitamin supplement,
N-acetylcysteine, a botanical supplement and bismuth tablets
(Table 3). Histopathologic examination of the original paraspinal
mass at the NIH confirmed a diffuse large B-cell lymphoma at stage
III, with a brisk mitotic rate.

Table 3

Patients with untreated stage III diffuse B-cell lymphoma have a
dismal prognosis. This case, like the preceding one, is unusual in
that the patient refused chemotherapy, which might have produced a
long-term remission. It appears, nonetheless, that a cure occurred
in connection with intravenous vitamin C infusions.

Discussion

These cases were analyzed in accordance with the NCI's Best Case
Series process, which reports and interprets apparent responses to
alternative therapies.23,24 Apart from its implications regarding
vitamin C, this article illustrates the use of the Best Case
Series approach in assessing the clinical plausibility of novel
therapies. None of these case histories provides definitive proof
that intravenous vitamin C therapy was responsible for the
patient's unusually favourable clinical course. It is often
difficult to prove definitively that a given treatment is
responsible for a specific clinical outcome. When the treatment is
unorthodox, alternative explanations, even if highly unlikely,
tend to be preferred.38,39 However, although they do not provide
grounds for advocating intravenous vitamin C therapy as a cancer
treatment, these cases increase the clinical plausibility of the
notion that vitamin C administered intravenously might have
effects on cancer under certain circumstances.

The overall plausibility of ascorbic acid administered
intravenously as a cancer therapy is enhanced by recent insights
into clinical pharmacokinetics and in vitro cancer-specific
cytotoxicity of vitamin C.15–20 Pharmacokinetics data show that
orally administered vitamin C results in tightly controlled plasma
and cell concentrations. Subjects consuming 200–300 mg per day of
vitamin C in 5 or more daily servings of fruits and vegetables
have fasting steady state plasma concentrations of about 70–80
μmol/L.40,41 Even with maximally tolerated oral doses of 3 g every
4 hours, peak plasma concentrations are estimated to not exceed
220 μmol/L.15 Intravenous administration of vitamin C bypasses
tight control for several hours, until homeostasis is restored by
renal excretion. Depending on the dose and infusion rate, peak
plasma concentrations obtained intravenously are estimated to
reach 14 000 μmol/L, and concentrations above 2000 μmol/L may
persist for several hours. Emerging in vitro data show that
extracellular ascorbic acid selectively kills some cancer but no
normal cells by generating hydrogen peroxide.20 Death is mediated
exclusively by extracellular ascorbate, at pharmacologic
concentrations that can be achieved only by intravenous
administration. Vitamin C may serve as a pro-drug for hydrogen
peroxide delivery to extravascular tissues, but without the
presence of hydrogen peroxide in blood. These data are consistent
with clinical pharmacokinetics of vitamin C administered
intravenously.15 Of note, only a minority of cancer patients
reported by Cameron and colleagues responded to intravenous and
oral vitamin C therapy,1–4 and not all cancer cells were killed by
ascorbic acid in vitro.20 Further basic investigation of
pharmacologic vitamin C concentrations in mediating cell death
will facilitate discovery of the mechanisms responsible for
sensitivity and resistance in vitro and in vivo. On the basis of
emerging clinical and in vitro data, early-phase clinical trials
of intravenous vitamin C therapy alone and in combination with
conventional chemotherapy are currently in the planning and
execution phase, including a formal phase I trial in progress at
McGill University.42–44

The cases reported here do not prove that vitamin C induced the
favourable outcomes observed. These patients received other
alternative medicine therapies. Spontaneous remission of some
tumours may occur rarely, although the 3 cancers reported here are
dissimilar. Accretion of more cases meeting NCI Best Case Series
guidelines may indicate whether vitamin C or other factors
contribute to such remissions.

It is likely that high vitamin C intakes have low toxicity, except
under certain conditions.45,46 Intravascular hemolysis was
reported after massive vitamin C administration in people with
glucose-6-phosphate dehydrogenase deficiency.46 Administration of
high-dose vitamin C to patients with systemic iron overload may
increase iron absorption and represents a contraindication.46,47
Ascorbic acid is metabolized to oxalate, and 2 cases of acute
oxalate nephropathy were reported in patients with pre-existing
renal insufficiency given massive intravenous doses of vitamin
C.48,49 Therefore, patients with renal insufficiency or renal
failure, or who are undergoing dialysis, should not receive high
doses of vitamin C.46 It is controversial whether high-dose
vitamin C use is associated with oxalate kidney stones, and
patients with hyperoxaluria or a prior history of oxalate kidney
stones have a relative contraindication to high-dose vitamin C.46
Rare cases of acute tumour hemorrhage and necrosis were reported
in patients with advanced cancer within a few days of starting
high-dose intravenous vitamin C therapy, although this was not
independently verified by pathologic review.1,50 Although tumour
hemorrhage suggests an anticancer potential for ascorbate, there
is the potential for risk to some patients.

The cases reported here are of tumours confirmed by
histopathologic examination to have poor prognosis but that
instead had long clinical remissions. Most previous case reports
lacked independent pathologic confirmation of the tumour and did
not follow the NCI Best Case Series guidelines, which makes their
interpretation difficult. Recent findings show that only high-dose
intravenous, but not oral, vitamin C therapy results in very high
plasma vitamin C concentrations (e.g., 14 000 μmol/L). At these
concentrations, the vitamin is toxic to some cancer cells,
possibly because at these concentrations the vitamin is a pro-drug
for hydrogen peroxide formation in extracellular fluid.
Accumulated data confer some degree of biological and clinical
plausibility to the notion that high-dose intravenous vitamin C
therapy may have anti-tumour effects in certain cancers. When all
available data are considered, further clinical study as to safety
and efficacy of intravenous vitamin C is warranted.

Acknowledgments

The writing of this paper was supported in part by the Intramural
Research Program of the National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health (Z01
DK 54506).

FootnotesEditor's take

• Intravenous administration of the maximum tolerated dose of
vitamin C produces plasma levels 25 times that achieved when the
same dose is administered orally. At high plasma concentrations
vitamin C is toxic to some cancer cells but not to normal cells in
vitro.

• Using the National Cancer Institute Best Case Series guidelines,
the authors reviewed 3 cases of advanced cancer where patients had
unexpectedly long survival times after receiving high-dose
intravenous vitamin C therapy.

Implications for practice: In a setting of biological plausibility
and clinical plausibility, further research into vitamin C as a
treatment for cancer is warranted.

Supplemental ascorbate in the
supportive treatment of cancer: Prolongation of survival
times in terminal human cancer.

E Cameron and L Pauling

Abstract

Ascorbic acid metabolism is associated with a number of mechanisms
known to be involved in host resistance to malignant disease.
Cancer patients are significantly depleted of ascorbic acid, and
in our opinion this demonstrable biochemical characteristic
indicates a substantially increased requirement and utilization of
this substance to potentiate these various host resistance
factors. The results of a clinical trial are presented in which
100 terminal cancer patients were given supplemental ascorbate as
part of their routine management. Their progress is compared to
that of 1000 similar patients treated identically, but who
received no supplemental ascorbate. The mean survival time is more
than 4.2 times as great for the ascorbate subjects (more than 210
days) as for the controls (50 days). Analysis of the survival-time
curves indicates that deaths occur for about 90% of the
ascorbate-treated patients at one-third the rate for the controls
and that the other 10% have a much greater survival time,
averaging more than 20 times that for the controls. The results
clearly indicate that this simple and safe form of medication is
of definite value in the treatment of patients with advanced
cancer.

Supplemental ascorbate in the
supportive treatment of cancer: Reevaluation of prolongation
of survival times in terminal human cancer

Ewan Cameron and Linus Pauling

AbstractA study has been made of the survival times of 100 terminal
cancer patients who were given supplemental ascorbate, usually 10
g/day, as part of their routine management and 1000 matched
controls, similar patients who had received the same treatment
except for the ascorbate. The two sets of patients were in part
the same as those used in our earlier study [Cameron, E. &
Pauling, L. (1976) Proc. Natl. Acad. Sci. USA 73, 3685-3689].
Tests confirm that the ascorbate-treated patients and the matched
controls are representative subpopulations of the same population
of “untreatable” patients. Survival times were measured not only
from the date of “untreatability” but also from the precisely
known date of first hospital attendance for the cancer that
eventually reached the terminal stage. The ascorbate-treated
patients were found to have a mean survival time about 300 days
greater than that of the controls. Survival times greater than 1
yr after the date of untreatability were observed for 22% of the
ascorbate-treated patients and for 0.4% of the controls. The mean
survival time of these 22 ascorbate-treated patients is 2.4 yr
after reaching the apparently terminal stage; 8 of the
ascorbate-treated patients are still alive, with a mean survival
time after untreatability of 3.5 yr.

New insights into the physiology and
pharmacology of vitamin CSebastian J. Padayatty and Mark Levine

Pharmacokinetic data are now available for the absorption, plasma
concentrations and renal excretion of vitamin C. With these new
insights, it is worth reconsidering the clinical role of vitamin C
in scurvy and subclinical deficiency, the amount of vitamin C
required for good health and, in particular, the speculative use
of high doses of vitamin C to treat cancer.

Scurvy, the dread of sailors past, conjures up images of perilous
sea voyages and rough tattooed men laid low by ignorance of a
vital amine, later designated vitamin C (which, as it happens, is
not an amine). While scurvy devastated seafarers, it was endemic
in the landbound, occurring widely wherever fruit and vegetables
were in short supply. Military campaigns from the Crusades to the
Napoleonic Wars, the American Civil War, and even World War I were
stymied by widespread and often fatal scurvy among the troops. The
antiscorbutic principle, identified and named ascorbic acid
(vitamin C) in 1932, is a simple water-soluble sugar-like
molecule. Early experiments, though perhaps flawed, showed that
the consumption of as little as 10 mg of vitamin C a day would
prevent signs of clinical scurvy.1 Although minute amounts will
forestall death, the optimum requirements for good health are not
known. Vitamin C is concentrated in many tissues, but these tissue
stores are easily depleted. James Lind's Treatise on Scurvy, first
published in 1753, reported the onset of the disease in sailors
after a month and a half at sea and described lassitude as its
early and invariable symptom.2 Depletion–repletion studies in
volunteers using a diet free of vitamin C have shown that plasma
vitamin C falls below 10 μmol/L in less than a month. At these
concentrations, fatigue is invariably present,3 and physical signs
appear soon after.1 A person who has been consuming 100 mg of
vitamin C daily will not develop scurvy for a month, even if the
intake of vitamin C is stopped altogether.3 Recently, on the
advice of the Food and Nutrition Board of the US National Academy
of Sciences, US and Canadian recommended dietary allowances were
increased from 60 mg per day to 75 mg per day for women and 90 mg
per day for men. Vitamin C intake is less than 60 mg in 20%–30% of
US adults. It is even lower among many population subgroups,
including children. Subclinical vitamin C deficiency is much more
common than is generally recognized,4 especially because the first
symptom of deficiency is fatigue, a nonspecific and common
complaint.

As an electron donor, vitamin C acts as a cofactor for 8 enzymes
involved in collagen hydroxylation, biosynthesis of carnitine and
norepinephrine, tyrosine metabolism and amidation of peptide
hormones.5 Vitamin C also has many nonenzymatic actions. It is a
powerful water-soluble antioxidant and, at physiological
concentrations, probably does not produce reactive intermediaries.
It protects low-density lipoproteins from oxidation, reduces
harmful oxidants in the stomach and promotes iron absorption. Its
antioxidant role in vivo is, however, unclear. Plasma ascorbic
acid concentrations may be low in chronic or acute oxidant states
such as in diabetes, in smokers, or following acute pancreatitis
or myocardial infarction. Ascorbic acid is easily oxidized to the
unstable dehydroascorbic acid. Dehydroascorbic acid is not
normally detectable in plasma but may occur transiently during
oxidant stress. Ascorbic acid is transported into the cell by
sodium-dependent vitamin C transporters SVCT1 and SVCT2, one or
both of which are found in most tissues.6 Dehydroascorbic acid is
transported by glucose transporters GLUT1 and GLUT3, and, in
insulin-sensitive tissues, also by GLUT4. When exposed to
bacteria, neutrophils oxidize extracellular ascorbic acid to form
dehydroascorbic acid, which is transported into the neutrophil and
rapidly reduced to ascorbic acid by the protein glutaredoxin (Fig.
1). As a result of this recycling of extracellular ascorbic acid,
the neutrophil internal concentration of ascorbic acid increases
10-fold.7 Ascorbic acid may quench oxidants generated during
phagocytosis and, thus, protect the neutrophil and surrounding
tissues from oxidative damage. Brain, adrenal cortex, liver,
spleen, pancreas and kidney tissues concentrate vitamin C for
unknown reasons.

When given orally, ascorbic acid is well absorbed at lower doses,
but absorption decreases as the dose increases. Thus, median
bioavailability following an oral dose is 87% for 30 mg, 80% for
100 mg, 72% for 200 mg and 63% for 500 mg. Less than 50% of a
1250-mg dose is absorbed, and most of the absorbed dose is
excreted in the urine.3,8 Ascorbic acid is not protein bound, so
it is filtered and reabsorbed by the kidneys in healthy subjects
but is lost in patients who have been hemodialyzed. Ascorbic acid
begins to appear in urine at doses above 100 mg/day, corresponding
to a plasma concentration of about 60 μmol/L, at which point
plasma is 70% saturated and circulating white blood cells are
fully saturated. Decreased bioavailability and renal excretion
keep plasma vitamin C at less than 100 μmol/L, even with an oral
dose of 1000 mg. In men at steady state, a 30-mg daily intake
results in a mean plasma concentration of 9 μmol/L, 60 mg results
in25 μmol/L, 100 mg in 56 μmol/L and 200 mg in 75 μmol/L. Thus,
the dose–concentration relationship is sigmoidal, with the steep
portion of the curve lying between 30 mg and 100 mg of oral
vitamin C daily.3,8 Doses greater than 500 mg daily contribute
little to plasma or tissue stores. Circulating white blood cells
contain 10–30 times the plasma concentrations of vitamin C.

In addition to the physiological role of ascorbic acid, it may
have unrelated pharmacological effects. When ascorbic acid is
administered intravenously, the limiting absorptive mechanism is
bypassed and very high plasma levels are attained. Following the
administration of 1.25 g intravenously, a peak plasma level of
1000 μmol/L is reached, even though 100 μmol/L is not exceeded by
oral dosing.8 When 5–10 g is given intravenously, the resulting
plasma levels may be as high as 5000 μmol/L.9

This difference between the oral and intravenous administration of
high doses was not adequately appreciated in studies of the
treatment of cancer with vitamin C. In vitro, ascorbic acid is
cytotoxic to many malignant cell lines10 at concentrations that
can be achieved in plasma by intravenous, but not oral,
administration. Whether similar effects would occur in vivo is not
known. The unconventional studies of Cameron and Campbell,11 later
joined by Linus Pauling, used high-dose intravenous vitamin C to
treat terminal cancer. They reported clinical benefits and
improved survival.12,13 Because these studies were not randomized
or placebo controlled, Moertel and colleagues carried out 2
randomized placebo-controlled clinical trials at the Mayo Clinic
to check these findings.14,15 Using high-dose oral vitamin C, they
found no benefit. Given the recent appreciation of the differing
plasma levels resulting from oral versus intravenous
administration, it is difficult to compare the studies carried out
by Moertel and coworkers with those of Cameron and his colleagues.
The cause of cancer patients will be better served if advocates
and sceptics concerning the efficacy of vitamin C re-examine these
issues with both open minds and scientific rigour.

We now know that plasma vitamin C concentrations are tightly
controlled and that the vitamin is concentrated by many tissues.
The optimum intake of vitamin C, its function in various tissues
and its antioxidant actions in vivo remain to be elucidated. In
the meantime, we should rigorously explore the potential
anticancer effects of vitamin C, when administered intravenously
at high doses, in patients with well-documented cancer16 in whom
other options have been exhausted. If these studies show promise,
then randomized clinical trials should follow.

Footnotes

Clinicians who wish to submit cases for review can contact Drs.
Padayatty and Levine for instructions at the address below.

Human pharmacokinetics data indicate that i.v. ascorbic acid
(ascorbate) in pharmacologic concentrations could have an
unanticipated role in cancer treatment. Our goals here were to
test whether ascorbate killed cancer cells selectively, and if so,
to determine mechanisms, using clinically relevant conditions.
Cell death in 10 cancer and 4 normal cell types was measured by
using 1-h exposures. Normal cells were unaffected by 20 mM
ascorbate, whereas 5 cancer lines had EC50 values of <4 mM, a
concentration easily achievable i.v. Human lymphoma cells were
studied in detail because of their sensitivity to ascorbate (EC50
of 0.5 mM) and suitability for addressing mechanisms.
Extracellular but not intracellular ascorbate mediated cell death,
which occurred by apoptosis and pyknosis/necrosis. Cell death was
independent of metal chelators and absolutely dependent on H2O2
formation. Cell death from H2O2 added to cells was identical to
that found when H2O2 was generated by ascorbate treatment. H2O2
generation was dependent on ascorbate concentration, incubation
time, and the presence of 0.5-10% serum, and displayed a linear
relationship with ascorbate radical formation. Although ascorbate
addition to medium generated H2O2, ascorbate addition to blood
generated no detectable H2O2 and only trace detectable ascorbate
radical. Taken together, these data indicate that ascorbate at
concentrations achieved only by i.v. administration may be a
pro-drug for formation of H2O2, and that blood can be a delivery
system of the pro-drug to tissues. These findings give
plausibility to i.v. ascorbic acid in cancer treatment, and have
unexpected implications for treatment of infections where H2O2 may
be beneficial.

Ascorbic acid (vitamin C, ascorbate) has a controversial history
in cancer treatment (1). Observational reports described
ascorbate, given in pharmacologic doses of 10 g daily, as
effective in treating some cancers and in improving patient
well-being (2-4). Subsequently, the same dose had no effect on
patient well-being and survival in two double-blind
placebo-controlled trials, and ascorbate was discarded as a
treatment modality (5, 6). Recent clinical evidence, however,
indicates that the role of ascorbate in cancer treatment should be
examined anew (7). The originally reported observational studies
used i.v. and oral ascorbate, but the subsequent double-blind
placebo-controlled studies used only oral ascorbate. It was not
recognized that the route of ascorbate administration might
produce large differences in plasma concentrations. Recent
pharmacokinetics studies in men and women show that 10 g of
ascorbate given i.v. is expected to produce plasma concentrations
of nearly 6 mM, which are >25-fold higher than those
concentrations from the same oral dose (7-9). As much as a 70-fold
difference in plasma concentrations is expected between oral and
i.v. administration, depending on dose. Despite inconsistencies,
some in vitro studies showed that ascorbate killed cancer cells,
although mechanisms and physiologic relevance were unclear
(10-12). Complementary and alternative medicine practitioners
worldwide currently use ascorbate i.v. in some patients, in part
because there is no apparent harm (13-15).

Given its potential safety and benefit, there is merit in
investigating i.v. ascorbate as a possible novel cancer treatment
modality. It is essential first to learn whether ascorbate acts as
an anticancer agent in vitro, and if so, by what mechanisms. Our
goals were to address the following: Does ascorbate in
pharmacologic concentrations kill cancer cells, but not normal
cells, using conditions that mimic i.v. use and a clinically
relevant time course? Is action dependent on extracellular
ascorbate, intracellular ascorbate, or both? If effective, what
are the mechanisms? Can ascorbate be delivered to tissues without
harm? Are there implications for other diseases?

We studied ascorbate at physiologic (0.1 mM) and pharmacologic
(0.3-20 mM) concentrations using 1-h incubations to mimic clinical
i.v. use (7-9). The data showed that pharmacologic concentrations
of ascorbate killed cancer but not normal cells, that cell death
was dependent only on extracellular but not intracellular
ascorbate, and that killing was dependent on extracellular
hydrogen peroxide (H2O2) formation with ascorbate radical as an
intermediate. Ascorbate generated detectable levels of H2O2 in
extracellular medium in the presence of trace serum protein but
not in whole blood. The findings indicate that ascorbate at
pharmacologic concentrations in blood may be a pro-drug for H2O2
delivery to tissues, with major therapeutic implications.

Materials and Methods

Cells and Reagents. Human Burkitt's lymphoma cells (JLP-119) were
obtained and studied as described in ref. 16. Other cell lines
were purchased from American Type Culture Collection and were
grown at 37°C in 5% CO2/95% air in recommended media containing
10% FBS (GIBCO). Human lymphocytes and monocytes were isolated by
apheresis (17) from at least six healthy subjects and used
immediately. Ascorbic acid was always buffered to pH 7.0 with
sodium hydroxide and prepared immediately before use.
Dehydroascorbic acid was freshly prepared (18).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
was purchased from Molecular Probes and bacto-agar was from Difco.
Other reagents, enzymes, and media were from general commercial
sources.

Cell Death. Nuclear staining with Hoechst 33342 (Hoechst
Pharmaceuticals) and propidium iodide (PI) was used for
morphological assessment of apoptosis, necrosis, and
pyknosis/necrosis by fluorescence microscopy as described in ref.
19. Briefly, 2.5 × 105 cells per ml were incubated with ascorbate
or H2O2 for 1 h, washed with PBS, and suspended in fresh media.
After 18-22 h, at least 200 cells were stained with Hoechst/PI and
visualized under fluorescence microscopy.

MTT was used as a screening assay and performed as described in
ref. 20. Cells in 96-well plates were treated with ascorbate
(0.1-20 mM) for 1 h, washed, and incubated for an additional 24 h.
The EC50 value was the concentration that reduced survival by 50%.

For colony formation on soft agar plates, cells were treated with
5 mM ascorbate for 1 h, washed, and plated. A two-layer agar
system was used, and colonies were visualized after 10-14 days
(21).

To determine the effects of red blood cells on ascorbate-induced
cell death, red blood cells were prepared by centrifugation of
heparinized human blood at 500 × g for 30 min. Human Burkitt's
lymphoma cells at 2.5 × 105 cells per ml were mixed with red blood
cells, 25% or 50% hematocrit. Cell mixtures were treated with 2 mM
ascorbate for 1 h. Lymphoma cells were recovered by using
Vacutainer CPT tubes (Becton Dickinson) according to the
manufacturer's instructions. After washing, lymphoma cells were
returned to fresh medium and assessed after 18 h by nuclear
staining as above.

Quantitative Procedures. Catalase activity was determined by using
Amplex Red (Molecular Probes) (22). Glutathione was detected by
using 5,5′-dithio bis-2-nitrobenzoic acid, and glutathione
peroxidase activity was measured by a coupled reaction with
glutathione reductase (Cayman Chemical, Ann Arbor, MI), according
to the manufacturer's instructions.

Ascorbate was measured by HPLC with coulometric electrochemical
detection (26). Protein was determined by using bicinchoninic acid
(27). Cell volumes were determined by using a Coulter Multisizer
II cell counter. Intracellular ascorbate concentrations were
calculated by converting cell protein to a measured intracellular
volume (18).

Results

Effects of Ascorbic Acid in Pharmacologic Concentrations on
Survival of Tumor and Normal Cells. We first investigated whether
ascorbate in pharmacologic concentrations selectively affected the
survival of cancer cells by studying nine cancer cell lines, four
normal cell types, and clinically relevant conditions. Clinical
pharmacokinetics analyses show that pharmacologic concentrations
of plasma ascorbate, from 0.3 to 15 mM, are achievable only from
i.v. administration (7). These concentrations are cleared within
hours by renal filtration and excretion. In contrast, plasma
ascorbate concentrations from maximum possible oral doses cannot
exceed 0.22 mM because of limited intestinal absorption, which is
bypassed with i.v. administration (7-9). To mimic potential
clinical i.v. use, tested cells were incubated for 1 h with either
pharmacologic ascorbate concentrations (0.3-20 mM) or a high
physiologic concentration (0.1 mM) as control. Once ascorbate was
removed, cell survival was determined by nuclear staining or MTT
after 24 h (Fig. 1A). For five of the nine cancer cell lines,
ascorbate concentrations causing a 50% decrease in cell survival
(EC50 values) were less than 5 mM, a concentration easily
achievable from i.v. infusion (7). All tested normal cells were
insensitive to 20 mM ascorbate.

Fig. 1.
Effects of pharmacologic ascorbic acid concentrations on cancer
and normal cells. Concentrations in this and all figures indicate
final concentrations. (A) EC50 values of ascorbate in human and
mouse cancer cells and normal human cells. All cells were ...

Colony formation assays were used as an additional means to
determine cell survival (21). Four cancer cell lines were
incubated with 5 mM ascorbate or untreated media for 1 h. Cells
were diluted and plated and growth assessed after 14 days (Fig.
1B). All four untreated cell lines grew in soft agar, whereas
three of four exposed to ascorbate displayed at least 99% growth
inhibition.

Effects of Ascorbic Acid on Death of Human Lymphoma Cells. Human
lymphoma cells (JLP-119) were studied in detail to determine the
effects of ascorbate on cell death. Lymphoma cells were selected
because of their sensitivity to ascorbate (Fig. 1A), the
suitability of these cells for nuclear staining to characterize
the mode of cell death (16, 19, 28), and the report of a positive
clinical response of lymphoma to i.v. ascorbate (14) (unpublished
work). Cells were incubated for 1 h with 0.1-5 mM ascorbate and
washed, and Hoechst/PI nuclear staining was performed 18 h later
to determine the amount and type of cell death (Fig. 2A).
Ascorbate induced concentration-dependent cell death, which was
nearly 100% at 2 mM. As ascorbate concentration increased, the
pattern of death changed from apoptosis to pyknosis/necrosis, a
pattern suggestive of H2O2-mediated cell death (19). We determined
the time necessary for cell death after exposure to 2 mM ascorbate
for 1 h (Fig. 2B). Apoptosis occurred by 6 h after exposure, and
cell death by pyknosis was ≈90% at 14 h after exposure. In
contrast to lymphoma cells, there was little or no killing of
normal lymphocytes and monocytes by ascorbate (Fig. 2C).

Fig. 2.
Effects of ascorbic acid on human Burkitt's lymphoma cells. Cells
were treated for 1 h, washed, and recultured without ascorbate.
Amounts and types of cell death were determined 18-22 h later by
nuclear staining with Hoechst/PI. Types of cell death: necrosis
...

The roles of intracellular versus extracellular ascorbate in
causing cell death were examined, using ascorbate and its oxidized
product dehydroascorbic acid. Ascorbate is transported into cells
as such by sodium-dependent transporters, whereas dehydroascorbic
acid is transported into cells by glucose transporters and then
immediately reduced internally to ascorbate (29). By using either
external ascorbate or external dehydroascorbic acid, lymphoma
cells were loaded to equal internal concentrations of ascorbate
over 1 h (data not shown). Despite similar intracellular ascorbate
concentrations under both conditions, cells died only when
ascorbate was present externally (Fig. 2D).

Similar to most cultured cells, lymphoma cells contain no
ascorbate unless the vitamin is added to the extracellular medium
(data not shown) (17). In contrast, excepting red blood cells, all
cells in vivo or acutely isolated contain ascorbate, usually in
millimolar concentrations. We investigated whether the prior
presence of intracellular ascorbate affected death mediated by
extracellular ascorbate. Lymphoma cells were preloaded with
physiologic concentrations of ascorbate to produce millimolar
intracellular concentrations, similar to normal lymphocytes (8,
9). Their response to external ascorbate was compared with
unloaded cells (Fig. 2E). Whether or not intracellular ascorbate
was preloaded, extracellular ascorbate induced the same amount and
type of death. Taken together, the data in Fig. 2 A-E indicate
that extracellular ascorbate in pharmacologic concentrations
mediates death of lymphoma cells by apoptosis and
pyknosis/necrosis, independently of intracellular ascorbate.

Mechanism of Ascorbate-Mediated Cell Killing. To determine the
mechanism of ascorbate-mediated lymphoma cell death, we tested the
effects of the membrane-impermeant H2O2-scavenger catalase, the
membrane-permeant H2O2-scavenger tetrakis (4-benzoic acid)
meso-substituted manganoporphyrin (MnTBAP) (30), and the
thiol-reducing agent Tris (2-carboxyethyl) phosphine hydrochloride
(TCEP) (31). We also tested whether adventitious transition metals
were responsible, by using the membrane impermeant chelator
diethylenetriamine-pentaacetic acid (DTPA) (32) and the membrane
permeant chelator
N,N′-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED)
(33-35) (Fig. 3A). The H2O2 scavengers were completely protective,
identifying H2O2 as the effector species mediating pharmacologic
ascorbate-induced cell death. The effect of ascorbate was not due
to chelatable, trace redox-active metals, because the two
chelators had no effect on preventing death. Superoxide dismutase
was not protective (data not shown), consistent with its action in
producing but not degrading H2O2 (36).

Because these data implicated H2O2 in cell killing, we added H2O2
to lymphoma cells and studied death patterns using nuclear
staining (19, 28). The death patterns found with exogenous H2O2
exposure were similar to those found with ascorbate. For both
ascorbate and H2O2, death changed from apoptosis to
pyknosis/necrosis as concentrations increased (Fig. 3B).

As a specific test of ascorbate action, the amount of H2O2 formed
in the presence of ascorbate was measured by using an oxygen
electrode. We compared the effects on cell death of H2O2 amounts
formed in the presence of ascorbate to effects from exogenously
added H2O2. H2O2 generated by ascorbate oxidation and exogenously
added H2O2 produced cell death curves that were indistinguishable
(Fig. 3C).

Sensitivity to direct exposure to H2O2 was greater in lymphoma
cells compared with normal lymphocytes and normal monocytes (Fig.
3D), consistent with the cytotoxicity pattern found above with
pharmacologic ascorbate exposure. Taken together, these data are
consistent with the conclusion that extracellular ascorbate
induced cell death by formation of H2O2.

We investigated whether activities of intracellular H2O2-removal
systems correlated with ascorbate-mediated cell death, for all
cells studied. There was no association between the EC50 for
ascorbate-mediated cell death and intracellular glutathione
concentrations, catalase activity, or glutathione peroxidase
activity (data not shown).

Mediators and Inhibitors of H2O2 Generation. H2O2 concentrations
generated by ascorbate were similar with tumor cells, normal
cells, or in medium without cells (data not shown), as measured by
using an oxygen electrode as above. H2O2 generation was dependent
on time, ascorbate concentration, and the presence of trace
amounts of serum in media (Fig. 4 A and B).

Based on these data, the most cogent explanation of ascorbate
action in forming H2O2 is that the first step is ascorbate
oxidation to its radical. We measured H2O2 concentration as a
function of ascorbate radical concentration and found a linear
relationship (Fig. 4C). These data imply that ascorbate radical is
a surrogate marker for H2O2 formation.

For ascorbate to be useful clinically, it should increase the
steady-state concentration of H2O2 in the extracellular milieu but
not in blood. We predicted that steady-state concentrations of
H2O2 generated by ascorbate oxidation would be undetectable in
blood for several reasons. First, if any ascorbate radical is
generated in blood, only very low concentrations are expected, and
such concentrations should be lower than that needed to form
detectable steady-state concentrations of H2O2 (37). Second,
whatever H2O2 is generated should be removed by glutathione
peroxidase and catalase within red blood cells, because H2O2 is
membrane permeable (38-41). These predictions were explored in the
following experiments. First, ascorbate (0-10 mM) was added to
whole blood and to medium, and ascorbate radical was measured by
electron paramagnetic resonance. Ascorbate radical in whole blood
was not detectable when ascorbate concentrations were <3 mM and
was present at minimal concentrations thereafter. In contrast,
there was robust ascorbate radical generation in medium, a
surrogate for extracellular fluid (Fig. 5A). Second, as direct
tests, H2O2 concentrations were measured under the following
conditions: In whole blood in the presence of varying
concentrations of ascorbate, in whole blood after exogenous H2O2
addition, and in medium with varying concentrations of ascorbate
(Fig. 5B). H2O2 was not detected in whole blood under either
condition, even in the presence of far higher added concentrations
than could be generated by ascorbate oxidation. Control formation
of H2O2 as a function of ascorbate concentration in medium
proceeded as expected. These data indicate that even if ascorbate
radical was formed in blood and H2O2 was generated, it would be
immediately scavenged to concentrations below detection limits.
Based on these data, an additional functional experiment was
conducted, based on the prediction that blood would protect tumor
cells from ascorbate-mediated cell death. Lymphoma cells were
incubated in the presence or absence of red blood cells, with and
without added ascorbate. Red blood cells completely protected
lymphoma cells from ascorbate-mediated cell death (Fig. 5C). Taken
together, these data indicate that ascorbate cannot generate
sustainable H2O2 concentrations in whole blood. The data are
consistent with the hypothesis that ascorbate in pharmacologic
concentrations is a pro-drug for H2O2 generation in the
extracellular milieu but not in blood.

Fig. 5.
Human blood inhibits H2O2 and ascorbate radical generation from
ascorbate. Ascorbate radical was measured by electron paramagnetic
resonance, H2O2 was measured by oxygen electrode, and cell death
was measured and displayed as in Fig. 2. (A) Ascorbate ...

Discussion

Our data show that ascorbic acid selectively killed cancer but not
normal cells, using concentrations that could only be achieved by
i.v. administration and conditions that reflect potential clinical
use. The effect was due only to extracellular and not
intracellular ascorbate, consistent with clinical i.v. dosing.
Ascorbate-mediated cell death was due to protein-dependent
extracellular H2O2 generation, via ascorbate radical formation
from ascorbate as the electron donor. Like glucose, when ascorbate
is infused i.v., the resulting pharmacologic concentrations should
distribute rapidly in the extracellular water space (42). We
showed that such pharmacologic ascorbate concentrations in media,
as a surrogate for extracellular fluid, generated ascorbate
radical and H2O2. In contrast, the same pharmacologic ascorbate
concentrations in whole blood generated little detectable
ascorbate radical and no detectable H2O2. These findings can be
accounted for by efficient and redundant H2O2 catabolic pathways
in whole blood (e.g., catalase and glutathione peroxidase)
relative to those in media or extracellular fluid (38-41). The
totality of the data are consistent with the interpretation that
ascorbic acid administered i.v. in pharmacologic concentrations
may serve as a pro-drug for H2O2 delivery to the extracellular
milieu, but without H2O2 accumulation in blood.

Although it is possible that H2O2 might accumulate in blood, this
would occur only under specific conditions that reflect on the
general safety of i.v. ascorbate. Ascorbate administered i.v. is
likely to be safe in most patients, with virtually no toxicity
compared to most currently available cancer chemotherapeutic
agents. The occurrence of one predicted complication, oxalate
kidney stones, is controversial (13). In patients with
glucose-6-phosphate dehydrogenase deficiency, i.v. ascorbate is
contraindicated because it causes intravascular hemolysis (13).
The mechanism of this previously unexplained observation is now
straightforward, based on the results here. H2O2 generated in
blood is normally removed by catalase and glutathione peroxidase
within red blood cells, with internal glutathione providing
reducing equivalents. The electron source for glutathione is NADPH
from the pentose shunt, via glucose-6-phosphate dehydrogenase. If
activity of this enzyme is diminished, the predicted outcome is
impaired H2O2 removal causing intravascular hemolysis, the
observed clinical finding.

Ascorbate as a potential cancer therapeutic agent has a
controversial and emotionally charged past (1, 3-6). Clinical
observational studies reported possible benefit in selected
patients, but double-blind placebo-controlled studies reported no
benefit, and ascorbate was discarded as a potential therapy by
conventional practitioners. Only recently has it been understood
that the discordant clinical findings can be explained by
previously unrecognized fundamental pharmacokinetics properties of
ascorbate (7). In vitro effects of ascorbate on death and survival
of cell lines have been reported, but there are multiple
experimental concerns. For example, reports compared an
experimental condition to that with no ascorbate at all (43, 44),
but such a condition has had unclear physiologic relevance,
because ascorbate outside and inside cells is always present
unless there is severe scurvy. It was unclear whether observed
effects were due to extracellular or intracellular ascorbate, or
both (12, 43-46). Some experiments have used widely varying
incubation times and ascorbate concentrations that have had no
corresponding clinical context, making interpretation difficult.
H2O2 generation by ascorbate oxidation in culture media was
variously interpreted as artifact (47, 48), even though chelators
had no effect (49), or reported to mediate damage internally due
to diminished intracellular ascorbate, but using an H2O2 assay in
which ascorbate could interfere (43, 44).

The experiments presented here provide a clear clinical context
for ascorbate action. Conditions were selected to reflect peak
ranges of i.v. ascorbate concentrations, which clinically might
last a few hours at most, depending on the infusion rate (7).
Intracellular transport of ascorbate is tightly controlled in
relation to extracellular concentration (8, 9, 29). Intravenous
ascorbate infusion is expected to drastically change extracellular
but not intracellular concentrations (8, 9). For i.v. ascorbate to
be clinically useful in killing cancer cells, pharmacologic but
not physiologic extracellular concentrations should be effective,
independent of intracellular ascorbate concentrations. This was
what was observed here. The experiments here provide a cohesive
explanation for ascorbate action in generating H2O2 outside cells,
without H2O2 accumulation in blood, leading to the conclusion that
ascorbate at pharmacologic concentrations in blood is a pro-drug
for H2O2 delivery to tissues.

We observed that H2O2 generation was independent of metal
chelators and dependent on at least 0.5% extracellular protein.
The responsible proteins were between 10 and 30 kDa (data not
shown). It is reasonable that extracellular milieu contains these
proteins, given that extracellular milieu protein is as much as
20% of serum protein, and favors lower-molecular-weight proteins
(50). Although identities of the proteins responsible are unknown,
we postulate that they may have redox-active metal centers. While
chelators may marginally affect these metals, they could
participate in the oxidation of ascorbate when it is at
pharmacologic concentrations, with subsequent formation of
superoxide and H2O2 (34). It is also possible that in vivo, cell
membranes and their associated proteins could harbor metals
accessible to extracellular fluid and could react similarly. In
either case, ascorbate, an electron-donor in such reactions,
ironically initiates pro-oxidant chemistry and H2O2 formation (34,
51).

It is unknown why ascorbate, via H2O2, killed some cancer cells
but not normal cells. There was no correlation with
ascorbate-induced cell death and glutathione, catalase activity,
or glutathione peroxidase activity. The data here showed that
ascorbate initiated H2O2 formation extracellularly, but H2O2
targets could be either intracellular or extracellular, because
H2O2 is membrane permeant (38, 52). For example, extracellular
H2O2 might target membrane lipids, forming hydroperoxides or
reactive intermediates that are quenched or repaired in normal
cells but not in sensitive cancer cells. In sensitive but not
resistant cancer cells, intracellular H2O2 could target DNA, DNA
repair proteins, or mitochondria because of diminished superoxide
dismutase activity (53). New insights may follow from future
studies of a very broad range of tumor cells or from microarray
analysis of resistant and sensitive cells derived from the same
genetic lineage.

H2O2, as the product of pharmacologic ascorbate concentrations,
has potential therapeutic uses in addition to cancer treatment,
especially in infections. H2O2 is a potent mammalian antimicrobial
defense mechanism (54). Neutrophils generate H2O2 from superoxide,
in turn formed by NADPH oxidase-catalyzed reduction of molecular
oxygen. There may be particular therapeutic application in
patients with chronic granulomatous disease who have diminished
superoxide production (55). Old observational animal experiments,
although uncontrolled, suggest that i.v. ascorbate is effective in
some viral infections (56, 57). This finding is also consistent
with in vitro experiments, in which H2O2 is toxic to hepatitis C
(58). Use of ascorbate as an H2O2-delivery system against
sensitive pathogens, viral or bacterial, has substantial clinical
implications that deserve rapid exploration.

To proceed clinically in potential treatment of infectious
diseases and cancer, clear safety documentation of i.v. ascorbate
administration is necessary. More than 100 patients have been
described, presumably without glucose-6-phosphate dehydrogenase
deficiency, who received 10 g or more of i.v. ascorbate with no
reported adverse effects other than tumor lysis (3, 4, 15, 59).
However, these descriptions lack formal safety documentation.
Complementary and alternative medicine practitioners worldwide
currently use ascorbate i.v. in doses as high as 70 g over several
hours (14, 15, 59). Because i.v. ascorbate is easily available to
people who seek it, a phase I safety trial in patients with
advanced cancer is justified and underway.

Acknowledgments

This work was supported in part by the Intramural Research
Programs of the National Institute of Diabetes and Digestive and
Kidney Diseases and the National Cancer Institute (National
Institutes of Health Grant Z01 DK 54506).

The debate that has been taking place in CMAJ about alternative
cancer therapies is extremely valuable, especially when considered
against the backdrop of intense public interest in this
subject.1,2,3,4,5,6 Like it or not, government agencies and
medical research centres are willing to evaluate alternative
therapies, and money from public and private sources is available
to pay the costs. The paradox, of course, is that by definition
unconventional, unorthodox or complementary therapies — whatever
one's favourite term may be — lack scientific credibility. How,
then, does one select the most promising of them for evaluation,
and what assessment procedures will be regarded as sufficiently
thorough and definitive by mainstream medical scientists,
government agencies and the proponents of alternative therapy?

Practitioners of alternative medicine and community physicians
can, no doubt, form successful partnerships with the aim of
providing high-quality, holistic care. Many mainstream Canadian
physicians already integrate elements of alternative therapy into
their practices. Alternative and Western science–based medicine
are either partners or amicable neighbours in most parts of the
world.

But it would be a triumph of hope over experience to assume that
alternative therapists and clinical researchers will easily
develop partnerships to test alternative cancer therapies. This
situation has to change because each side needs to learn from the
other. The alternative therapists need expertise in rigorous
documentation and the principles of clinical investigation, as
well as the physical and intellectual resources necessary to
design and conduct informative clinical trials. Clinical
researchers need information about the specific aims of a given
alternative therapy, insight into how it is used and the
perspective necessary to design pragmatic trials that assess it
fairly. The effort should be made to identify genuinely promising
new approaches and to explore, rather than refute, them.7,8,9
Young scientific investigators may be disinclined to travel this
rocky road. My own dean — now retired — encouraged my interest in
alternative medicine, but added, “Keep your day job.”

It was with such considerations in mind that I, with Dr. Carmen
Tamayo and Dr. Mary Ann Richardson of the University of Texas
Center for Alternative Medicine Research, organized a 2-day
research workshop in Montreal last spring to consider the
background and current status of the most luminously controversial
of all biological, alternative cancer therapies, high-dose vitamin
C. Our workshop brought together mainstream physicians, research
oncologists, alternative practitioners and a representative of the
US Food and Drug Administration. Funding was provided by the Lotte
and John Hecht Memorial Foundation, a charitable organization
endowed in British Columbia in 1962 with a major objective of
supporting the investigation of alternative and complementary
therapies, especially for cancer. A selection of some of the
workshop presentations has appeared elsewhere,10,11,12,13 and a
full summary report is in preparation, but it is appropriate to
relate here a powerful personal lesson from this experience.

The vitamin C and cancer controversy began in 1974 with the
publication, by Cameron and Campbell, of a careful description of
the responses of 50 consecutive patients with advanced,
untreatable cancer to high-dose intravenous and oral vitamin C.14
Most patients did not respond, but extraordinary things happened
to a significant minority of them. These included several tumour
regressions and 4 cases (2 of them fully documented at autopsy) of
catastrophic tumour hemorrhage and necrosis occurring within 3–6
days after starting vitamin C therapy. Cameron had never seen
anything like this in his long and distinguished career as an
oncologic surgeon, and he concluded that an important phenomenon
was occurring that merited further investigation. Two-time Nobel
laureate Linus Pauling championed the cause. As a result of the
ensuing controversy, a double-blind controlled clinical trial of
10 g/day oral vitamin C was carried out at the Mayo Clinic in
patients with a variety of untreatable, terminal cancers.15 The
results were negative. Pauling and Cameron objected that, unlike
in Scotland, all the Minnesota patients had received prior
cytotoxic therapy and that this could have mitigated the
restorative biological effects of vitamin C.

A second clinical trial was carried out in patients with
colorectal cancer at an earlier stage than in the first trial, but
for which the only cytotoxic therapy then available, fluorouracil,
was known to be ineffective and hence could ethically be
withheld.16 The results of this trial were also negative, but
Pauling and Cameron objected to the way that it was designed and
carried out. A patient's course of vitamin C (or placebo) was
terminated as soon as there was clear evidence that his or her
tumour was continuing to progress, whereas Cameron and Pauling's
key claim was that vitamin C prolongs the life of cancer patients
when given continuously. Indeed, they had previously emphasized
the danger of withdrawing vitamin C abruptly, having found this to
be associated with a rebound acceleration of the disease. They
also objected to the lack of effort to ensure compliance with the
study medication or to screen the control group for illicit use of
vitamin C.17,18 Another obvious problem was the lack of
statistical power. Cameron had found that no more than a minority
of patients responded in the prompt and dramatic fashion typical
of cytotoxic drugs, yet the latter was the type of response the
Mayo Clinic trial was designed to detect. It is interesting to
note that the year in which the second Mayo Clinic trial was
published, the New England Journal of Medicine also published, to
wide acclaim, the clinical responses of an uncontrolled series of
cancer patients treated with the biological agent, interleukin-2,
at the US National Cancer Institute (NCI).19 As with Cameron's
Scottish patients, only a minority of the patients responded. Had
interleukin-2 been assessed as vitamin C was in the Mayo Clinic
study, it, too, might well have been found to be ineffective.

In 1989 Pauling visited the head of the NCI, Samuel Broder, and
described cases of what he claimed were complete cancer remissions
in response to vitamin C. Broder was sufficiently interested to
convene an NCI panel to review 25 case histories of patients to be
selected by Cameron as providing plausible evidence that high-dose
vitamin C could have important biological effects in human cancer.
The cases selected included 2 complete remissions experienced by a
patient with stage IV non-Hodgkin's lymphoma following courses of
vitamin C therapy,20,21 the disappearance of multiple brain
lesions diagnosed as metastatic on clinical grounds and CT
scanning in a patient with bronchogenic carcinoma, tumour
regression in a patient with metastatic renal cell carcinoma, and
autopsy-confirmed tumour hemorrhage in a patient within 3 days of
initiating vitamin C therapy.18 In 1991 Pauling received a letter
informing him of the panel's conclusion that vitamin C had not
been shown to be responsible for improved outcome in any cancer
case, either because the cancer diagnosis was not sufficiently
proven or because an explanation other than vitamin C therapy
might have accounted for the patient's clinical course. In some
cases, extraordinarily long survivals were not credited to vitamin
C because of lack of information about such long survivals in the
natural history of the disease.

When submitting their case histories, Cameron and Pauling
understood that they would be evaluated with regard to the
plausibility of the hypothesis that vitamin C could have important
biological effects in human cancer. Instead, considering each case
separately from all the others, the NCI panel looked for proof
that vitamin C must have been responsible for the clinical effects
reported and exact confirmation, not plausibility, of the tissue
diagnosis. Proof is necessary to change medical practice,
plausibility to justify testing a clinical hypothesis. Neither
side in this exchange was wrong, but it would have been helpful if
they had understood each other's position.

The lesson to be learned from this is that the parameters of the
debate about alternative therapies — the “rules of engagement” —
between mainstream cancer researchers and proponents of
alternative therapy need to be clearly defined and the goals must
be explicit and common to both parties. To do otherwise leads to
the risk of unintended confusion and heightening of the barrier of
mistrust that already stands between many individuals involved in
this debate. Proponents of alternative therapy have an obligation
to provide grounds for biological plausibility, such as sound
theoretical or preclinical data, or for clinical plausibility, in
the form of authentic, well-prepared case reports, in order to
justify the investment of time and energy in exploring the merits
of a novel anticancer therapy. But plausibility, not proof, should
be sufficient to initiate the process.

Since the Mayo Clinic trials were published, rational guidelines
for testing biological agents like vitamin C have been
developed,22 and new information has emerged since the NCI review
took place about the biological effects and clinical
pharmacokinetics of vitamin C.18,23,24 In this issue (page 353),25
Sebastian Padayatty and Mark Levine (Levine was also a member of
the NCI panel that reviewed the cases submitted by Pauling and
Cameron) describe these new developments in our understanding of
vitamin C biology and their relevance to the question of a role
for vitamin C in cancer therapy. Perhaps it is time to revisit the
issue of the clinical and biological plausibility of a role for
vitamin C in cancer therapy.

Prevention and treatment of occlusive
cardiovascular disease with ascorbate and substances that
inhibit the binding of lipoprotein (A)US5278189

RATH MATTHIAS; PAULING LINUS

A method is provided for prevention and treatment of
cardiovascular disease, such as atherosclerosis, by administering
therapeutically effective dosages of a drug comprised of
ascorbate, lipoprotein(a) binding inhibitors, and antioxidants

DescriptionTECHNICAL FIELD

The present invention relates generally to the prevention and
treatment of cardiovascular disease and more particularly to
methods and compounds that inhibit the binding of lipoprotein (a)
to components of the arterial wall.

Essentially all human blood contains lipoprotein(a); however,
there can a thousand-fold range in its plasma concentration
between individuals. High levels of Lp(a) are associated with a
high incidence of cardiovascular disease. Armstrong, V. W., et al.
(1986) Atherosclerosis 62: 249-257; Dahlen, G., et al. (1986)
Circulation 74: 758-765; Miles, L. A., et al. (1989) Nature 339:
301-302; Zenker, G., et al. (1986). Stroke 17: 942-945 (The term
occlusive cardiovascular disease will be used hereafter as
including all pathological states leading to a narrowing and/or
occlusion of blood vessels throughout the body, but particularly
atherosclerosis, thrombosis and other related pathological states,
especially as occurs in the arteries of the heart muscle and the
brain.)

For some time, general medical practice has focused on the role of
LDL, the so called "bad cholesterol," in occlusive cardiovascular
disease. A great many studies have been published ostensibly
linking occlusive cardiovascular disease with elevated levels of
LDL. As a result, most therapies for the treatment and prevention
of arteriosclerosis rely on drugs and methods for the reduction of
serum levels of LDL's. Such therapies have had mixed results. The
efficacy of such approaches to the problem of occlusive
cardiovascular disease continues to be major source of debate.

There exists therefore a need for a drug therapy for reducing the
binding of Lp(a) to vessel walls, for reducing the overall level
of Lp(a) in the circulatory system and for promoting the release
of existing deposits of Lp(a) on vessel walls.

SUMMARY OF THE INVENTION

The foregoing needs in the treatment and prevention of
cardiovascular disease are met by the methods and compositions of
the present invention.

A method is provided for the treatment of occlusive cardiovascular
disease, comprising the step of administering to a subject an
effective amount of ascorbate and one or more binding inhibitors,
as a mixture or as a compound comprising ascorbate covalently
linked with binding inhibitors, which inhibit the binding of Lp(a)
to blood vessel walls, such as arterial walls. This effect may
also be obtained by administering an effective amount of one or
more inhibitors, without ascorbate. The term binding inhibitor
throughout the specification and claims is intended to include all
substances that have an affinity for the lysine binding site
present on the interior walls of blood vessels, particularly
arteries, the site of Lp(a) binding. Most of these substances
compete with plasmin for the lysine binding site and some of these
compounds, in high doses, are in clinical use for the treatment of
hyperfibrinolytic states.

A method is further provided for the prevention of atherosclerosis
comprising the step of administering to a subject an effective
amount of ascorbate and one or more binding inhibitors as
previously discussed but further comprising one or more
antioxidants. The term antioxidant throughout the specification
and the claims is intended to exclude ascorbate which has as one
of its chemical properties a potent antioxidant effect.

It is thus an object of the invention to provide a method for
treatment of occlusive cardiovascular disease by administering to
a subject an effective amount of ascorbate and one or more binding
inhibitors, or an effective amount of one or a mixture of binding
inhibitors.

It is another object of the invention to provide a method for
preventing of occlusive cardiovascular disease, by administering
to a subject an amount of ascorbate effective to lower the amount
of Lp(a) in the plasma of the subject.

Yet another object of the present invention is to provide a method
for prevention of cardiovascular disease by administering to a
subject an effective amount of ascorbate and one or more binding
inhibitors, or an effective amount of one or more binding
inhibitors.

A further object of the present invention is to provide a
pharmaceutically acceptable agent for the treatment of occlusive
cardiovascular disease.

Still another object of the present invention is to provide a
pharmaceutically acceptable agent for the prevention of
cardiovascular disease.

These and other objects will be more readily understood upon
consideration of the following detailed descriptions of
embodiments of the invention and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 is an immunoblot of the plasma of guinea pigs form
from the test described in Example 1.FIG. 2A a photograph of the aorta of a guinea pig receiving
an adequate amount of ascorbate from the test diet in Example 1.FIG. 2B is a photograph of an aorta of a guinea pig
receiving a hypoascorbic diet after three weeks from the test
diet in Example 1.FIG. 3 is an immunoblot of plasma and tissue of guinea pigs
from the test shown in Example 2.FIG. 4 shows the potential mechanism of binding inhibitors
in the therapy for atherosclerosis.FIG. 5 show the potential mechanism of ascorbate in the
binding of Lp(a) to the arterial wall.

DETAILED DESCRIPTION OF THE INVENTION

Our invention is based in part on our discovery that animals which
have lost the ability to produce ascorbate, such as higher
primates and guinea pigs, uniformly produce Lp(a). Most animals
which possess the ability to synthesize ascorbate generally do not
produce Lp(a). Further, we have found that ascorbate deficiency in
humans and guinea pigs tends to raise Lp(a) levels and causes
atherosclerosis by the deposition of Lp(a) in the arterial wall,
from which we conclude that ascorbate administration lowers plasma
Lp(a) levels.

We have also discovered that substances that inhibit the binding
of Lp(a) to components of the arterial wall, particularly to
fibrinogen, fibrin and fibrin degradation products herein
identified as binding inhibitors, such as lysine or
.epsilon.-aminocaproic acid used alone or in combination with
ascorbate, cause release of Lp(a) from the arterial wall. Thus,
ascorbate and such binding inhibitors are not only useful for the
prevention of occlusive cardiovascular disease, but also for the
treatment of such disease. The present invention, then, provides
methods and pharmaceutical agents for the both the treatment and
prevention of occlusive cardiovascular disease in vivo.

GENERAL APPLICATIONS

The present invention provides a method and pharmaceutical agent
for the treatment and prevention of occlusive cardiovascular
disease generally, by administering to a subject an effective
amount of ascorbate and one or more binding inhibitors. Throughout
the specification and claims, the term binding inhibitor is
intended to cover any substance which has as at least one of its
chemical properties the ability to inhibit the binding of Lp(a) to
blood vessel wall components, particularly to fibrin or
fibrinogen. As used herein, the term "ascorbate" includes any
pharmaceutically acceptable salt of ascorbate, including sodium
ascorbate, as well as ascorbic acid itself. Binding inhibitors
include, but are not limited to .epsilon.-aminocaproic acid,
lysine, tranexamic acid (4-aminomethylcyclohexane carboxylic
acid), p-aminomethylbenzoic acid, p-benzylamine sulfuric acid,
o-N-acetyl-lysine-methyl ester, PROBUCOL (a compound comprised of
2 butyl hydroxy tocopherol groups linked together by a disulphide
group), Aprotinin, trans-4-aminomethylcyclohexanecarboxylic acid
(AMCA), and benzamidine derivatives such as amidinophenylpyruvic
acid (APPA) and
1-naphthyl-(1)-3-(6-amidinonaphthyl-(2))-propanone-1 HCl (NANP).
An effective amount of a binding inhibitor or a mixture of binding
inhibitors may also be used, without ascorbate. Other substances
used in the treatment of occlusive cardiovascular disease may be
co-administered, including antioxidants, such as tocopherol,
carotene and related substances; vitamins; provitamins; trace
elements; lipid-lowering drugs, such as hydroxy-methyl-glutaryl
coenzyme A reductase inhibitors, nicotinic acid, fibrates, bile
acid sequestrants; and mixtures of any two or more of these
substances.

Although ascorbate can be used alone or in varying combinations
with one or more representative constituents of the above classes
of compounds, we prefer when treating a pre-existing
cardiovascular condition to combine ascorbate with at least one
each of the binding inhibitors, antioxidants and lipid lowering
drugs elements in the dosages (per kilogram of body weight per day
(Kg BW/d)) provided in Table 1. It should be noted that Table 1
provides differing concentration ranges of each constituent,
depending upon whether the agent is to be administered orally or
parenterally. The variance in dosages is reflective of variation
in disease severity. It will be realized therefore that if the
subject has been diagnosed for advanced stages of atherosclerosis,
dosages at the higher end of this range can be utilized. However,
if only prevention of an atherosclerosis condition is the object,
dosages at the lower end of this range can be utilized.

As an alternative, a pharmaceutical agent identical to the one
just described, but omitting ascorbate, may be employed.

Where ascorbate and binding inhibitors are utilized in the same
agent, they may simply be mixed or may be chemically combined
using synthesis methods well known in the art, such as compounds
in which ascorbate and the inhibitor are covalently linked, or
form ionically bound salts. For example, ascorbate may be bound
covalently to lysine, other amino acids, or .epsilon.-aminocaproic
acid by ester linkages. Ascorbyl .epsilon.-aminocaproate is such
an example. In this form the ascorbate moiety may be particularly
effective in preventing undesirable lipid peroxidation.

In the case of oral administration, a pharmaceutically acceptable
and otherwise inert carrier may be employed. Thus, when
administered orally, the active ingredients may be administered in
tablet form. The tablet may contain a binder such as tragacanth,
corn starch or gelatin; a disintegrating agent, such as alginic
acid, and/or a lubricant such as magnesium stearate. If
administration in liquid form is desired, sweetening and/or
flavoring agents may be used. If administration is by parenteral
injection, in isotonic saline, a phosphate buffered solution or
the like, may be used as a pharmaceutically acceptable carrier.

The advisability of using binding inhibitors in treating occlusive
cardiovascular disease will depend to some extent on the subject's
general health, particularly with regard to hyperfibrinolytic
conditions. Most binding inhibitors (except lysine) are used
clinically to treat such conditions. As a result, monitoring of
the subject's coagulation and fibrinolytic sysem is recommended
before and during treatment for occlusive cardiovascular disease.
Long-term administration of binding inhibitors will require
formulations in which the dosages of binding inhibitors are in the
lower ranges of the dosages given in Table 1.

Prevention, as contrasted with treatment, of cardiovascular
disease may be accomplished by oral or parenteral administration
of ascorbate alone. Table 1 gives a range of ascorbate
concentrations sufficient to lower the serum Lp(a) concentration.

Preferably the prevention of the occlusive cardiovascular disease
according to the invention is accomplished by use of a physical
mixture of ascorbate and one or more binding inhibitors, or by use
of a compound comprising covalently linked ascorbate with one or
more of the binding inhibitors, which inhibit binding of Lp(a) to
the arterial wall. A binding inhibitor or mixture of binding
inhibitors may also be administered without ascorbate to prevent
Lp(a)-associated occlusive cardiovascular disease.

To optimize the therapeutic effect of the release of Lp(a) from
the blood vessel walls, the ascorbate and the binding inhibitors
described above may be separately administered. Further
optimization of therapeutic effect can be gained by using a time
release composition to achieve relatively constant serum
concentrations of the agent through time.

TABLE 1DOSAGES OF COMPONENTS IN THE DRUG COMPOSITIONS OF THE
PRESENT INVENTIONOral Parenteral Administration

EXPERIMENTAL

Having disclosed the preferred embodiment of the present
invention, the following examples are provided by way of
illustration only and are not intended to limit the invention in
any way.

EXAMPLE 1

Because of its metabolic similarity to man, with resepct to the
metabolism of ascorbate and Lp(a), the guinea pig was used in this
example.

No study has been previously reported in the guinea pig to
identify the lopoprotein involved as risk factors in plasma and as
constituents of the atherosclerotic plaque.

Three female Gartly guinea pigs with an average weight of 800 g
and an approximate age of 1 year wer stuided. One animal received
an extreme hypoascorbic diet with 1 mg ascorbate/kg body weight/d.
Another animal received 4 mg/kg BW/d. The third animal served as a
control receiving 40 mg ascorbate/kg/BW/d)

Blood was drawn by heart puncture from the anesthetized animals
and collected into EDTA containing tubes at the beginning, after
10 days, and after 3 weeks, when the animals were sacrificed.
Plasma was stored at -80 DEG C. until analyzed. Lp(a) was detected
in the plasma of the guinea pigs by use of SDS-polyacrylamide gels
according to Neville (J. Biol. Chem., 246, 6328-6334 (1971))
followed by Western blotting (Beisiegel, et al., J. Biol. Chem.,
257, 13150-13156 (1982)). 40 .mu.l of plasma and 20 mg of arterial
wall homogenate were applied in delipidated form per lane of the
gel. The immunodetection of apo(a) was performed using a
polyclonal anti-human apo(a) antibody (Immuno, Vienna, Austria)
followed by a rabbit anti-sheep antibody (Sigma) and the
gold-conjugated goat anti-rabbit anti-body with subsequent silver
enhancement (Bio-Rad). The determinations of cholesterol and
triglycerides were done at California Veterinary Diagnostics
(Sacramento) using the enzyme assay of Boehringer Mannheim. Plasma
ascorbate was determined by the dinitrophenylhydrazine method
(Schaffer, et al., J. Biol. Chem., 212, 59 (1955)).

Vitamin C deficiency in the diet led to an increase of Lp(a) in
the plasma of the guinea pig indicated by a clear band in the
immunoblot of the plasma after 10 and 20 days of a hypoascorbic
diet (FIG. 1). At necropsy the animals were anesthetized with
metophase and were exsanguinated. Aorta, heart and various other
organs were taken for biochemical and histological analysis. The
aorta was excised, the adventitial fat was carefully removed, and
the vessel was opened longitudinally. Subsequently the aorta was
placed on a dark metric paper and a color slide was taken. The
picture was projected and thereby magnified by an approximate
factor 10. The circumference of the ascending aorta, the aortic
arch and thoracic aorta as well as the atherosclerotic lesions in
this area were marked and measured with a digitalized planimetry
system. The degree of atherosclerosis was expressed by the ratio
of plaque area to the total aortic area defined. The difference in
the 3 one-year old animals of the experiment was significant and
pronounced lesions were observed in the ascending aorta and the
arch of the vitamin C deficient animal (FIG. 2B).

EXAMPLE 2

To confirm the data obtained in Example 1, a second guinea pig
experiment was conducted, using 33 male animals with a mean weight
of 550 g and an approximate age of 5 months. One group of 8
animals served as a control and received 40 mg ascorbate/kg BW/d
(group A). To induce hypoascorbemia 16 animals were fed 2 mg
ascorbate/kd/d (group B). Group A and half of the animals of group
B (progression sub-group) were sacrificed after 5 weeks as
described above. Half of group B was kept for 2 more weeks,
receiving daily intraperitoneal injection of 1.3 g sodium
ascorbate/Kg BW/d as a daily intra peritoneal injection with the
intention to reduce the extent of atherosclerosis lesions. After
this period these animals also were sacrificed.

The aortas of the guinea pigs receiving a sufficient amount of
ascorbate were essentially plaque free, with minimal thickening of
the intima in the ascending region. In contrast, the
ascorbate-deficient animals exhibited fatty streak-like lesions,
covering most parts of the ascending aorta and the aortic arch. In
most cases the branching regions of the intercostal arteries of
the aorta exhibited similar lipid deposits. The difference in the
precentage of lesion area between the control animals and the
hypoascorbic diet animals was 25% deposition of lipids and
liporpoteins in the arterial wall.

TABLE 3MEAN PLASMA PARAMETERS OF THE DIFFERENT GROUPS IN RELATION
TO THE AREA OF AORTIC LESIONS

EXAMPLE 3

Human arterial wall was obtained post mortem from the aorta
ascendens. The tissue showed homogeneous intimal thickening (early
atherosclerotic lesion). It was cut into pieces, with 100 mg of
the cut up tissue homogenized in a glass potter for a 1 minute in
2.5 ml of the following solutions:

Results of this treatment are given in Table 4 and show that,
compared to the control solution, a considerable amount of Lp(a)
was released from the interior arterial wall.

TABLE 4Lp(a) RELEASED FROM HUMAN AORTA IN RELATION TO SPECIFIC
BINDING INHIBITORS

By now it is apparent that the methods and compositions of
the present invention meet longstanding meeds in the field of
prevention and treatment of occlusive cardiovascular disease.
Although preferred embodiments and examples have been disclosed,
it is understood that the invention is in no way limited by them,
but rather is defined by the claims that follow and equivalents
thereof.

Use of ascorbate and tranexamic acid
solution for organ and blood vessel treatment prior to
transplantationUS5230996

RATH MATTHIAS; PAULING LINUS

A method and pharmaceutical agent are provided for the prevention
and treatment of cardiovascular disease, particularly
cardiovascular disease in the context of diabetic angiopathy,
by-pass surgery, organ transplantation, and hemodialysis, by
administering ascorbate and substances that inhibit the binding of
lipoprotein (a) to blood vessel walls. The use of ascorbate and
lipoprotein (a) binding inhibitors such as tranexamic acid in a
temporary storage solution for blood vessels and organs prior to
transplantation is also demonstrated.

TECHNICAL FIELD

The present invention relates generally to the prevention and
treatment of cardiovascular disease arising as a complication from
surgery or a pre-existing, unrelated disease, and more
particularly to methods and compounds for that inhibit the binding
of lipoprotein (a) to components of the arterial wall.

Essentially all human blood contains lipoprotein (a); however,
there can a thousand-fold range in its plasma concentration
between individuals. High levels of Lp(a) are associated with a
high incidence of cardiovascular disease. Armstrong, V. W., et al.
(1986) Atherosclerosis 62: 249-257;Dahlen, G., et al. (1986)
Circulation 74: 758-765; Miles, L. A., et al. (1989) Nature 339:
301-302; Zenker, G., et al. (1986) Stroke 17: 942-945 (The term
cardiovascular disease will be used hereafter as including all
pathological states leading to a narrowing and/or occlusion of
blood vessels throughout the body, but particularly
atherosclerosis, thrombosis and other related pathological states,
especially as occurs in the arteries of the heart muscle and the
brain.)

It has also been suggested that Lp(a), the concentration of which
increases markedly in the blood during pregnancy, may be linked to
cardiovascular disease in woman. Zechner, R., et al. (1986)
Metabolism 35: 333-336. It has also been observed that diabetics,
many of whom suffer in some degree from atherosclerotic diseases,
display greatly elevated serum levels of Lp(a). Bruckert, E., et
al. (1990) JAMA 263: 35-36.

In addition to atherosclerosis and thrombosis in arteries Lp(a)
has also been linked to stenosis of vein grafts after coronary
bypass surgery. Hoff, H., et al. (1988) Circulation 77: 1238-1244.
Similar problems of rapid occlusion of vessels have been observed
in heart transplant patients.

For some time, general medical practice has focused on the role of
LDL, the so called "bad cholesterol," in cardiovascular disease. A
great many studies have been published ostensibly linking
cardiovascular disease with elevated levels of LDL. As a result,
most therapies for the treatment and prevention of
arteriosclerosis rely on drugs and methods for the reduction of
serum levels of LDLS. Such therapies have had mixed results. The
efficacy of such approaches to the problem of cardiovascular
disease continues to be major source of debate.

There exists a need, therefore, for a drug and therapy for
reducing the binding of Lp(a) to vessel walls, for reducing the
overall level of Lp(a) in the circulatory system and for promoting
the deposition of existing deposits of Lp(a) on vessel walls.

SUMMARY OF THE INVENTION

The foregoing needs in the treatment and prevention of
cardiovascular disease are met by the methods and compositions of
the present invention.

A method is provided for the treatment of cardiovascular disease,
particularly atherosclerosis, induced or promoted by kidney
failure, diabetes, transplant surgery and the like, comprising the
step of administering to a subject an effective amount of
ascorbate and one or more binding inhibitors, as a mixture or as a
compound comprising ascorbate covalently linked with binding
inhibitors, which inhibit the binding of Lp(a) to blood vessel
walls, such as arterial walls and vein grafts used in by-pass
surgery. This effect may also be obtained by administering an
effective amount of one or more inhibitors, without ascorbate. The
term binding inhibitor throughout the specification and claims is
intended to include all substances that have an affinity for the
lysine binding site present on the interior walls of blood
vessels, particularly arteries, the site of Lp(a) binding. Most of
these substances compete with plasmin for the lysine binding site
and some of these compounds, in high doses, are in clinical use
for the treatment of hyperfibrinolytic states.

A method is further provided for the prevention of atherosclerosis
related to or as a complication of surgery, a preexisting disease
or a therapy such as hemodialysis. comprising the step of
administering to a subject an effective amount of ascorbate and
one or more binding inhibitors as previously discussed but further
comprising one or more antioxidants. The term antioxidant
throughout the specification and the claims is intended to exclude
ascorbate, which itself is a powerful antioxidant.

It is thus an object of the invention to provide a method for
treatment of induced cardiovascular disease by administering to a
subject an effective amount of ascorbate and one or more binding
inhibitors, or an effective amount of one or a mixture of binding
inhibitors.

It is another object of the invention to provide a method for
prevention of induced cardiovascular disease, by administering to
a subject an amount of ascorbate effective to lower the amount of
Lp(a) in the plasma of the subject.

Yet another object of the present invention is to provide a method
for prevention of induced cardiovascular disease by administering
to a subject an effective amount of ascorbate and one or more
binding inhibitors, or an effective amount of one or more binding
inhibitors.

A further object of the present invention is to provide a
pharmaceutically acceptable agent for the treatment of induced
cardiovascular disease.

Still another object of the present invention is to provide a
pharmaceutically acceptable agent for the prevention of induced
cardiovascular disease.

Yet another object of the present invention is to provide a method
for preservation of explanted tissues and organs that reduces the
risk of occurrence of cardiovascular disease in the tissues and
organs after implantation.

It is also an object of the present invention to provide a
pharmaceutically acceptable agent to assist in the preservation of
explanted tissues and organs prior to implantation.

Still another object of the present invention is to provide a
pharmaceutical compound and method for treating cardiovascular
disease arising from a preexisting condition of diabetes mellitus.

These and other objects will be more readily understood upon
consideration of the following detailed descriptions of
embodiments of the invention and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1 is an immunoblot of the plasma of guinea pigs from
the test described in Example 1. Increase of Lp(a) in plasma of
guinea pigs with a hypoascorbic diet. Immuunoblot with anti
apo(a) antibodies. Lane 1: human control plasma. Lane 2: guinea
pig plasma at the start of experiment. Lane 3: guinea pig plasma
after 10 days of hypoascorbic diet. Lane 4; guinea pig plasma
after 20 days. HMW: high molecular weight standard.FIG. 2A is a photograph of the aorta of a guinea pig
receiving an adequate amount of ascorbate from the test diet in
Example 1.FIG. 2B is a photograph of an aorta of a guinea pig
receiving a hypoascorbic diet after three weeks from the test
diet in Example 1.FIG. 3 is an immunoblot of plasma and tissue of guinea pigs
from the test shown in Example 2. Plasma and tissue of guinea
pigs. Immunoblot with anti apo(a) antibody. HC: human control
plasma; L: liver tissue; B: brain tissue; A: aortic tissue,
homogenate of plaque area from FIG. 2B.FIG. 4 is a diagrammatic representation of the action of
Lp(a) binding inhibitors on Lp(a) to cause release of Lp(a) from
fibrin fibers of an arterial wall.FIG. 5 is a diagrammatic representation of the action of
ascorbate to prevent association and reassociation of Lp(a) to
an arterial wall.

DETAILED DESCRIPTION OF THE INVENTION

Our invention is based in part on our discovery that animals which
have lost the ability to produce ascorbate, such as higher
primates and guinea pigs, uniformly produce Lp(a), whereas most
animals which possess the ability to synthesize ascorbate
generally do not produce Lp(a). Further, we have found that
ascorbate deficiency in humans and guinea pigs tends to raise
Lp(a) levels and causes atherosclerosis by the deposition of Lp(a)
in the arterial wall, from which we conclude that ascorbate
administration lowers plasma Lp(a) levels.

We have also discovered that substances that inhibit the binding
of Lp(a) to components of the arterial wall, particularly to
fibrinogen, fibrin and fibrin degradation products herein
identified as binding inhibitors, such as lysine or
.epsilon.-aminocaproic acid. Thus, ascorbate and such binding
inhibitors are not only useful for the prevention of
cardiovascular disease, but also for the treatment of such
disease.

Some beneficial effects of ascorbate in the prevention and
treatment of cardiovascular disease have been established (see
FIG. 2). Our invention reveals the relation to and therapeutic use
of ascorbate for Lp(a), one of the most atherogenic lipoproteins,
directly related to the development of atherosclerotic plaques.
The beneficial effects of ascorbate suggest that ascorbate
therapies would be useful in a variety of situations where
occlusion of blood vessels by Lp(a) deposition is a problem. For
instance, ascorbate may be useful in transplantation of blood
vessels and whole organs, where a combination of tissue damage to
the transplant, such as by oxidation, and high serum Lp(a) in the
transplant recipient results in rapid occlusion of blood vessels
in the transplant. Ascorbate may also be useful in the area of
hemodialysis, where loss of ascorbate and other vitamins and trace
elements from the blood of hemodialysis patients can result in
increased serum levels of Lp(a) and thus increased risk of
cardiovascular disease. Finally, it appears that ascorbate alone
and in combination with binding inhibitors, specifically with
plasmin competitors, may be therapeutically useful for treatment
of the pathogenic effects of diabetes which is associated with
elevated serum concentrations of Lp(a).

The present invention provides methods and pharmaceutical agents
for both the treatment and prevention of cardiovascular disease in
vivo; methods and agents for the preservation of damage linked
vessel occlusion in explanted tissues and organs, as well as
methods and agents for the prevention of hemodialysis-linked
cardiovascular disease. Each of these embodiments is discussed in
turn below.

GENERAL APPLICATIONS

The present invention provides a method and pharmaceutical agent
for the treatment and prevention of cardiovascular disease
generally, particularly atherosclerosis, by administering to a
subject an effective amount of ascorbate and one or more binding
inhibitors which inhibit the binding of Lp(a) to blood vessel wall
components, particularly to fibrin or fibrinogen. As used herein,
the term "ascorbate" includes any pharmaceutically acceptable salt
of ascorbate, including sodium ascorbate, as well as ascorbic acid
itself. Binding inhibitors include, but are not limited to
.epsilon.-aminocaproic acid (EACA), lysine, tranexamic acid
(4-aminomethylcyclohexane carboxylic acid), p-aminomethylbenzoic
acid, p-benzylamine sulfuric acid, and
.alpha.-N-acetyl-lysine-methyl ester and PROBUCOL (a compound
comprised of 2 butyl hydroxy tocopherol groups linked together by
a disulphide group), Aprotinin,
trans-4-aminomethylcyclohexanecarboxylic acid (AMCA), and
benzamidine derivatives such as amidinophenylpyruvic acid (APPA)
and 1-naphthyl-(1)-3-(6-amidinonaphthyl-(2))-propanone-1 HCl
(NANP). An effective amount of a binding inhibitor or a mixture of
binding inhibitors may also be used, without ascorbate. Other
substances used in the treatment of cardiovascular disease may be
co-administered, including: antioxidants, such as tocopherol,
carotene and related substances; vitamins; provitamins; trace
elements; lipid-lowering drugs, such as hydroxy-methyl-glutaryl
coenzyme A reductase inhibitors, nicotinic acid, fibrates, bile
acid sequestrants; and mixtures of any two or more of these
substances.

Although ascorbate can be used alone or in varying combinations
with one or more representative constituents of the above classes
of compounds, we prefer when treating a pre-existing
cardiovascular condition to combine ascorbate with at least one
each of the binding inhibitors, antioxidants and lipid lowering
drugs elements in the dosages (per kilogram of body weight per day
(kg BW/d)) provided in Table 1. It should be noted that Table 1
provides differing concentration ranges of each constituent,
depending upon whether the agent is to be administered orally or
parenterally. The variance in dosages is reflective of variation
in disease severity. It will be realized therefore that if the
subject has been diagnosed for advanced stages of atherosclerosis,
dosages at the higher end of this range can be utilized. However,
if only prevention of an atherosclerosis condition is the object,
dosages at the lower end of this range can be utilized.

As an alternative, a pharmaceutical agent identical to the one
just described, but omitting ascorbate, may be employed.

Where ascorbate and binding inhibitors are utilized in the same
agent, they may simply be mixed or may be chemically combined
using synthesis methods well known in the art, such as compounds
in which ascorbate and the inhibitor are covalently linked, or
form ionically bound salts. For example, ascorbate may be bound
covalently to lysine, other amino acids, or .epsilon.-aminocaproic
acid by ester linkages. Ascorbyl .epsilon.-aminocaproate is such
an example. In this form the ascorbate moiety may be particularly
effective in preventing undesirable lipid peroxidation.

In the case of oral administration, a pharmaceutically acceptable
and otherwise inert carrier may be employed. Thus, when
administered orally, the active ingredients may be administered in
tablet form. The tablet may contain a binder such as tragacanth,
corn starch or gelatin; a disintegrating agent, such as alginic
acid, and/or a lubricant such as magnesium stearate. If
administration in liquid form is desired, sweetening and/or
flavoring agents may be used. If administration is by parenteral
injection, in isotonic saline, a phosphate buffered solution or
the like, may be used as pharmaceutically acceptable carrier.

The advisability of using binding inhibitors in treating
cardiovascular disease will depend to some extent on the subject's
general health, particularly with regard to hyperfibrinolytic
conditions. Most binding inhibitors (except lysine) are used
clinically to treat such conditions. As a result, monitoring of
the subject's coagulation and fibrinolytic system is recommended
before and during treatment for cardiovascular disease. Long-term
administration of binding inhibitors will require formulations in
which the dosages of binding inhibitors are in the lower ranges of
the dosages given in Table 1.

Prevention, as contrasted with treatment, of cardiovascular
disease may be accomplished by oral or parenteral administration
of ascorbate alone. Table 1 gives a range of ascorbate
concentrations sufficient to lower the serum Lp(a) concentration.
Preferably the prevention of the cardiovascular disease according
to the invention is accomplished by use of a physical mixture of
ascorbate and one or more binding inhibitors, or by use of a
compound comprising covalently linked ascorbate with one or more
of the binding inhibitors, which inhibit binding of Lp(a) to the
arterial wall. A binding inhibitor or mixture of binding
inhibitors may also be administered without ascorbate to prevent
Lp(a)-associated cardiovascular disease.

To optimize the therapeutic effect of the release of Lp(a) from
the blood vessel walls, the ascorbate and the binding inhibitors
described above may be separately administered. Further
optimization of therapeutic effect can be gained by using a time
release composition to achieve relatively constant serum
concentrations of the agent through time.

CORONARY BYPASS APPLICATIONS

As discussed above, recurrence of cardiovascular disease after
bypass surgery is a frequent problem. Physicians often observe
that the veins used to replace occluded arteries become rapidly
occluded themselves after implantation, often requiring the
patient to undergo successive surgical episodes to replace clogged
bypasses. While not wishing to be bound to any theory, we believe
that the rapid occlusion observed in many individual's results
from a combination of the patient's pre-existing elevated levels
of Lp(a) and injury to the bypass veins, during transplantation,
particularly as a result of oxidative damage during explantation.
This damage makes binding of Lp(a) to the vessel interior easier.
Further, Lp(a) has been detected in abundance in reoccluded
by-pass veins after coronary bypass surgery. See, Cushing, et al.
(1989) Atherosclerosis 9:593-603. Lp(a) is now known to be the
most significant factor for reocclusion of bypass veins. See,
Hoff, H, et al. (1988) Circulation 77:1238-1244. Thus, a further
embodiment of this invention includes using the pharmaceutical
agent of the present invention to lower the bypass patient's Lp(a)
before, during and after surgery while at the same using a
solution containing the agent to rinse and store the bypass veins
until such time as the veins are implanted into the recipient,
thereby reducing oxidative damage that can make Lp(a) binding more
likely after implantation.

The treatment protocols for the bypass patient generally follow
those described above for the treatment of pre-existing
cardiovascular disease. The composition of the pharmaceutical
agent will generally include ascorbate, one or more binding
inhibitors, one or more antioxidants and one or more lipid
lowering drugs as enumerated and in the dosages given in Table 1.
Of course, the level of dosage will depend on disease severity.
Further, the constituents of the agent can be combined just as
described above, can be administered either orally or parenterally
and can be combined with a pharmaceutically acceptable carrier.

TABLE 1DOSAGES OF COMPONENTS IN THE DRUG COMPOSITIONS OF THE
PRESENT INVENTIONParenteral / Oral Administration

Turning now to vessel treatment and storage, it is important to
provide an in vitro environment which minimizes vessel injury. We
conclude that vessel injury can be reduced by the addition of a
combination of ascorbate, binding inhibitors and antioxidants to
the solution in which the vessels are normally stored. A range of
effective concentrations of these constituents in solution is
given in Table 2. The general aspects of live vessel preservation
and storage prior to implantation are well known in the art.

TABLE 2CONCENTRATION OF COMPONENTS IN THE SOLUTION OF THE PRESENT
INVENTION

We have also found that the solution and method of the present
invention are effective in preventing cardiovascular disease from
occurring in transplanted organs that have been otherwise
successfully implanted in an organ recipient, particularly in the
case of the heart.

As with occlusion of transplanted veins after bypass surgery, a
transplanted heart free of any substantial arterial occlusion may
suffer accelerated atherosclerosis after implantation. We believe
that the mechanism described for occlusion of transplanted vessels
applies equally to the heart itself as a whole, namely that the
heart muscle itself, as well as the interiors of the arterial
walls become damaged, making the arteries of the heart more prone
to binding with Lp(a). Because the organ recipient often presents
elevated serum concentration of Lp(a), particularly after surgery
(see, Maeda, S. et al. (1989) Atherosclerosis 78: 145-150),
atherosclerosis can proceed at an accelerated rate.

Treatment follows along the same line as that described for bypass
surgery. Damage to the organ itself is minimized by placing the
organ in a solution containing a mixture of ascorbate, binding
inhibitors, and antioxidants in an otherwise standard storage
solution. Concentration ranges for the various components in the
final solution are given in Table 2. Because of the oxidative
cellular damage during extended periods of explantation, the
concentration of antioxidants should be in the higher range of
dosages disclosed in Table 1. The standard storage solution itself
is well known in the art. Storage of the organ in this solution
will tend to minimize damage to arterial walls, thereby providing
fewer places for Lp(a) to bind.

Of course, patient treatment is also desireable. If the organ
recipientsuffers from some degree of atherosclerosis at the time
of organ transplant, the protocol and drug described above
generally for the treatment of atherosclerosis should be employed.
If, however, the patient does not suffer from atherosclerosis, use
of the drug and protocol described above for prevention of
atherosclerosis is desired. In all cases, the lowest dosages of
ascorbate should be employed in the drug composition since
ascorbate has an immune stimulatory effect.

APPLICATIONS IN HEMODIALYSIS TREATMENT

It is well known that patients who suffered renal failure and
require regular dialysis treatment to cleanse the blood of
metabolic waste products are also at an increased risk for
cardiovascular disease. We believe that the reason for this may be
a depletion of ascorbate, vitamins in general and other essential
substances from the blood supply during the hemodialytic process.
As described more fully above, the loss of ascorbate would result
in greater injury to the interior of the artery walls over time
and may also result in the production of elevated Lp(a) levels in
the blood serum.

As can appreciated, the solution and method of the present
invention can be applied both to the patient and the hemodialysis
solution to prevent and control hemodialysis-related
cardiovascular disease. Turning first to the hemodialysis
solution, it is desired to add a combination of ascorbate, binding
inhibitors and antioxidants to the solution to produce
concentrations of these compounds in solution in the range of
concentrations provided in Table 2.

In order to achieve the best results, treatment of the dialysis
patient should be carried out in addition to modification of the
hemodialysis solution. Treatment should follow the drug and
protocols set forth in detail above for the treatment of a
preexisting atherosclerotic condition.

APPLICATIONS IN TREATMENT OF DIABETES

The composition and method of the present invention are also
useful in the treatment of the pathological effects of diabetes
mellitus. In diabetes mellitus, pathological charges in the
arteries frequently lead to clinical symptoms or complete failure
in various organs such as the kidney, eye and peripheral
circulation system. Therefore, one therapeutic focus in diabetes
mellitus is the treatment of diabetic angiopathy.

It appears that glucose competitively inhibits the physiologic
uptake of ascorbate in different cell systems of the body,
including the arterial wall. Kapeghian, et al. (1984) Life Sci.
34: 577. Such damage to arterial walls creates binding sites for
Lp(a). Further, Lp(a) has been found to be elevated in the blood
serum of diabetic patients. The atherogenic process is perhaps
therefore accelerated by the combination of damaged arteries and
elevated Lp(a). Therefore, we propose that ascorbate alone or in
combination with at least one binding inhibitor has therapeutic
value in treating diabetes-related atherosclerosis.

Thus, another embodiment of the present invention is the use of a
composition and method in treating the pathogenic effects of
diabetes mellitus, particularly with regard to atherosclerotic
conditions.

The treatment protocol involves the oral or parenteral
administration of a pharmaceutical composition comprised of
ascorbate, one or more binding inhibitors and one or more
antioxidants. Dosages for a course of treatment are provided in
Table 1. The dosage of ascorbate should preferably fall within the
higher range, thereby increasing its chance of cellular uptake in
the presence of high serum levels of glucose.

EXPERIMENTAL

Having disclosed the preferred embodiment of the present
invention, the following examples are provided by way of
illustration only and are not intended to limit the invention in
any way.

EXAMPLE 1

Because of its metabolic similarity to man, with respect to the
metabolism of ascorbate and Lp(a), the guinea pig was used in this
example.

No study has been previously reported in the guinea pig to
identify the lipoprotein involved as risk factors in plasma and as
constituents of the atherosclerotic plaque.

Three female Hartly guinea pigs with an average weight of 800 g
and an approximate age of 1 year were studied. One animal received
an extreme hypoascorbic diet with 1 mg ascorbate/kg body weight/d.
Another animal received 4 mg/k BW/d. The third animal served as a
control receiving 40 mg ascorbate/Kg BW/d.

Blood was drawn by heart puncture from the anesthetized animals
and collected into EDTA containing tubes at the beginning, after
10 days, and after 3 weeks, when the animals were sacrificed.
Plasma was stored at -80 DEG C. until analyzed. Lp(a) was detected
in the plasma of the guinea pigs by use of SDS-polyacrylamide gels
according to Neville (J. Biol. Chem., 246, 6328-6334 (1971))
followed by Western blotting (Beisiegel, et al., J.Biol. Chem.,
257, 13150-13156 (1982)). 40 .mu.l of plasma and 20 mg of arterial
wall homogenate were applied in delipidated form per lane of the
gel. The immunodetection of apo(a) was performed using a
polyclonal anti-human apo(a) antibody (Immuno, Vienna, Austria)
followed by a rabbit anti-sheep antibody (Sigma) and the
gold-conjugated goat anti-rabbit antibody with subsequent silver
enhancement (Bio-Rad). The determinations of cholesterol and
triglycerides were done at California Veterinary Diagnostics
(Sacramento) using the enzyme assay of Boehringer Mannheim. Plasma
ascorbate was determined by the dinitrophenylhydrazine method
(Schaffer, et al., J. Biol. Chem., 212, 59 (1955)).

Vitamin C deficiency in the diet led to an increase of Lp(a) in
the plasma of the guinea pig indicated by a clear band in the
immunoblot of the plasma after 10 and 20 days of a hypoascorbic
diet (FIG. 1). At necropsy the animals were anesthetized with
metophase and were exsanguinated. Aorta, heart and various other
organs were taken for biochemical and histological analysis. The
aorta was excised, the adventitial fat was carefully removed, and
the vessel was opened longitudinally. Subsequently the aorta was
placed on a dark metric paper and a color slide was taken. The
picture was projected and thereby magnified by an approximate
factor 10. The circumference of the ascending aorta, the aortic
arch and thoracic aorta as well as the atherosclerotic lesions in
this area were marked and measured with a digitalized planimetry
system. The degree of atherosclerosis was expressed by the ratio
of plaque area to the total aortic area defined. The difference in
the 3 one-year old animals of the experiment was significant and
pronounced lesions were observed in the ascending aorta and the
arch of the vitamin C deficient animal (FIG. 2B).

EXAMPLE 2

To confirm the data obtained in Example 1, a second guinea pig
experiment was conducted, using 33 male animals with a mean weight
of 550 g and an approximate age of 5 months. One group of 8
animals served as a control and received 40 mg ascorbate/kg BW/d
(group A). To induce hypoascorbemia 16 animals were fed 2 mg
ascorbate/kd/d (group B). Group A and half of the animals of group
B (progression sub-group) were sacrificed after 5 weeks as
described above. Half of group B was kept for 2 more weeks,
receiving daily intraperitoneal injection of 1.3 Na-ascorbate/kg
BW/d as a daily intra peritoneal injection with the intention to
reduce the extent of atherosclerosis lesions. After this period
these animals also were sacrificed.

The aortas of the guinea pigs receiving a sufficient amount of
ascorbate were essentially plaque free, with minimal thickening of
the intima in the ascending region. In contrast, the
ascorbate-deficient animals exhibited fatty streak-like lesions,
covering most parts of the ascending aorta and the aortic arch. In
most cases the branching regions of the intercostal arteries of
the aorta exhibited similar lipid deposits. The difference in the
percentage of lesion area between the control animals and the
hypoascorbic diet animals was 25% deposition of lipids and
lipoproteins in the arterial wall.

TABLE 3MEAN PLASMA PARAMETERS OF THE DIFFERENT GROUPS IN RELATION
TO THE AREA OF AORTIC LESIONS

A possible inhibitor may be identified first by adding molar
amounts of the possible inhibitor at a little larger, by
approximately 5 times, the amount of .epsilon.-aminocaproic acid
found in the earlier study. If, at this concentration, a possible
inhibitor is found to inhibit the agglutination, studies are made
at lower concentrations, to determine the concentration that has a
50% initiatory effect.EXAMPLE 3

Human arterial wall was obtained post mortem from the aorta
ascendens. The tissue showed homogenous intimal thickening (early
atherosclerotic lesion). It was cut into pieces, with 100 mg of
the cut up tissue homogenized in a glass potter for 1 minute in
2.5 ml of the following solutions:

Results of this treatment are given in Table 4 and show that,
compared to the control solution, a considerable amount of Lp(a)
was released from the interior arterial wall.

TABLE 4Lp(a) RELEASED FROM HUMAN AORTA IN RELATION TO SPECIFIC
BINDING INHIBITORS

By now it is apparent that the methods and compositions of the
present invention meet longstanding needs in the field of
prevention and treatment of induced cardiovascular disease.
Although preferred embodiments and examples have been disclosed,
it is understood that the invention is in no way limited thereby,
but rather is defined by the claims that follow and the
equivalents thereof.

US5639787Therapeutic method for the treatment of cancer

Inventor(s): RIORDAN NEIL H [US]; RIORDAN HUGH
D [US] +

A method of treating cancer in a patient by raising and
maintaining the concentration of ascorbic acid, or ascorbate, in
the patient's plasma to at least the level expected to be toxic to
an in vitro culture of cells of the type of cancer being treated,
the required plasma ascorbate levels being achieved and maintained
using long term intravenous infusions of large amounts of
ascorbate, with or without ascorbate cytotoxicity effectiveness
enhancing or tumor site delivery and absorption enhancing agents.

This web site was established in 1996. The methods described here
for treating heart disease were invented by the great American
scientist Linus Pauling, and his German associate Matthias Rath,
MD. These methods and protocol have been proven in practice over
the past twenty years. At the required high dosages, the protocol
is called The Pauling Therapy in Linus Pauling's honor.
Unfortunately, heart patients are not going hear about this safe
and apparently very effective treatment (even cure) for heart
disease from their doctors. -- Owen Fonorow

The leading killer in the United States, the condition that those
in medicine call heart disease, or occlusive cardiovascular
disease, is really a low-grade form of scurvy. This fact is
becoming increasingly more difficult for modern medicine to deny.

Chronic scurvy: Heart disease is a misnomer. The disease is
characterized by scab-like build-ups that slowly grow on the walls
of blood vessels. The underlying disease process reduces the
supply of blood to the heart and other organs resulting in angina
("heart cramp"), heart attack and stroke. The correct terminology
for this disease process is chronic scurvy, a sub-clinical form of
the classic vitamin C deficiency disease.

The true nature of the disease was identified in the early 1950s
by a Canadian team led by G. C. Willis, MD. This finding was
confirmed in the late 1980s, by the world's then leading
scientist, Linus Pauling, Ph.D.

Pauling alerted the world in lectures, in writing and on video
after he and his associates conducted experiments that confirmed
the Willis findings. To date, this alert has never made its way
into a mainstream media outlet. Moreover, cardiologists are
taught, and routinely tell their patients, that there is no
connection between vitamin C and heart disease, and also that
there is no value in vitamin C in amounts much higher than the
minuscule RDA.

From a scientific standpoint, if a medical doctor, or anyone,
tries to challenge the true nature of cardiovascular disease, they
must be able to cite experiments that refute the Pauling/Willis
chronic scurvy hypothesis. Such experiments have never been
published.

Its been twelve years since Pauling issued the alert. Pharmacology
professors Steven Hickey and Hilary Roberts in their recent book
ASCORBATE: The Science of Vitamin C (2004) document, incredibly,
that there have been no independent experiments published that
were designed to test the Pauling hypothesis (except one at much
lower doses that was conducted by Pauling’s close associate Dr.
Matthias Rath.)

We are aware of only one clinical study in humans that has been
carefully designed to test the Pauling high-dose hypothesis. The
study was performed in the UK, with 200 men, over a period of
three years, and the data confirmed Pauling’s theory and therapy.
Yet, so far, Dr. Kale Kenton’s study has not appeared in a medical
journal.

Will the giant pharmaceutical industry facing these facts survive
or will it collapse in 2005? The end of the suppression of vitamin
C will reveal the CODEX restrictions for what they really are, a
means to prop up an industry that has little reason to exist in
its present form. The public is beginning to realize that the
world’s most profitable industry is really a house of cards. Its
most profitable products are at best useless, at worst dangerous.
Prescription drugs beget more drugs. The secret that dooms Big
Pharma is that the best of health is achieved by taking high doses
of vitamin C and avoiding toxic prescription medications as if
your life depended on it.

History of the Great Suppression

The 700,000 people who die needlessly every year are those who
heed their cardiologists advice. The American Heart Association
estimates that 63 million Americans suffer cardiovascular disease.
More than one million undergo some form of heart operation, and
over 15 million are taking statin cholesterol lowering drugs on
the advice of their doctor. These popular statin drugs are known
to deplete CoQ10 and probably cause heart failure.

The pioneering research into the relationship between vitamin C
and heart disease began in the late 1940s, not long after the
structure of vitamin C was determined. Canadians doctors proved
that a vitamin C 'deficiency' causes the condition, commonly
called atherosclerosis. These doctors found that the condition
will arise in 100 per cent of vitamin C-deprived animal test
subjects that don't make their own vitamin C. Furthermore, these
Canadian pioneers demonstrated that vitamin C alone reverses
atherosclerosis in laboratory animals. [THE REVERSIBILITY OF
ATHEROSCLEROSIS, G. C. Willis, Canad. M. A. J., July 15, 1957, Vol
77., Pg 106-109 ]

The team performed similar studies in humans. The results, while
not conclusive, showed reversals of atherosclerotic plaques in one
third of the human subjects. Notably, these studies were of low
doses, no more than 1500 mg per day. [SERIAL ARTERIOGRAPHY IN
ATHEROSCLEROSIS, G. C. Willis, A. W. Light, W.S. Cow, Canad. M. A.
J. Dec 1954, Vol 71, 1954, p. 562-568 ]

The knowledge that heart disease is a form of scurvy has been
suppressed from the time that the first series of Willis articles
were published in the Canadian Medical Journal in the early 1950s.
Inexplicably, since the 1950s, no articles favorable to vitamin C
and its connection with atherosclerosis have appeared in a
reputable medical journal that is widely read by medical doctors.

Cardiologists to-be are taught that there is no relation between
vitamin C intake and heart disease, and that it is quackery to
suggest otherwise. These assertions seem justified because reports
of such studies are lacking. But, as vitamin C expert and
pharmacological professor Steve Hickey points out, every
cardiologist could have performed these studies on his/her own.

"Time has moved on and the medical profession has FAILED over the
past 50 years to produce the required experiments. The budget of
say the NIH alone is over 27 billion but over the past 50 years no
one has replicated the early vitamin C and heart disease research,
which could be done by almost any cardiologist from petty cash.

Since Pauling, and others, have promoted ascorbate as a cure for
heart disease, it seems silly that a potential cure for the worst
killer in the developed nations (atherosclerosis) has not been
refuted. To a scientist from any other discipline this lack of
interest would be bizarre.

Its a fact that the experiments have been done in animals and the
results show that ascorbate protects against atherosclerosis and
may reverse it. There is some additional evidence from human
studies that is consistent with this interpretation. So why have
the human studies not been performed? Or we may ask if they have
been performed, was the data withheld? The enemies of Pauling, as
well as the drug companies, would love to see the Pauling
hypothesis discredited. Why have the experiments not been
reported? -- Dr. Steve Hickey email correspondence, Dec 2004

The Lecture Video

In a 1992 lecture recorded on video, Linus Pauling explained the
reason atherosclerosis forms on the walls of arteries when vitamin
C is deficient. This leading scientist explained how a specific
form of cholesterol that compensates for low vitamin C causing
plaques and why his discovery of a rapid cure for chronic scurvy
includes the amino acid lysine. [*]

There is no doubt that the news of this cure has been suppressed,
otherwise most of the public would have learned that twice Nobel
prize winner Linus Pauling had suggested it. Millions are dying
needlessly for lack of disseminated knowledge of the Pauling
discovery which amounts to the suppression of it.

Is Linus Pauling's high-dose ascorbic acid and amino acid therapy
the cure for heart disease? The crime is that no one knows!

Pauling and his former associate, Dr. Matthias Rath, did their
part by running the experiments and attempting to publicize these
discoveries. Now it is up to other researchers in the medical
scientific community. If there is the mere chance that Drs.
Willis, Pauling and Rath are correct, it is truly criminal to fail
to run experiments under fair conditions. Apparently, medical
science is controlled by the drug industry. Even the United States
National Institutes of Health gives all appearances of being under
the control of the drug industry and it seems that not even
members of Congress can overcome this obstacle.

Gross Negligence at the National Institutes of Health (NIH)

In the years 1998 and 2002 the Vitamin C Foundation submitted
grant requests for government funding to study Dr. Pauling
theories. These requests were formally submitted to the new Office
of Alternative Medicine at the NIH.
[http://vitamincfoundation.org/NCCAMgrant]

The reasons for these requests were twofold: The Foundation sought
funding so that Linus Pauling's recommended therapy would be
fairly investigated in humans. All previously known tests have
been performed with less than adequate amounts of vitamin C. These
grant applications also put the United States government on notice
that Linus Pauling had in fact made the claim of an outright cure
for heart disease.

The NIH was free to design, sponsor and run their own study with
their own choice of scientists. Had such studies been conducted,
millions of lives and billions of dollars might now have been
saved. Unfortunately for Americans, the NIH, Office of Alternative
Medicine, rejected both grant requests, and failed to run their
own studies.

Both requests were turned down by the United States Government,
not because the reviewers had any objection to the science or the
protocols, but because the scientists and medical doctors that the
Foundation recruited to run the study were "inexperienced."
Apparently, investigators that run studies for the NIH have to be
members of "The Club." This travesty is a matter of public record.
- Mike Till, Sr.

The Cholesterol Lowering Drug Debacle

Cardiology has been on the wrong path for a long time. The result
has been that heart disease is still the leading cause of
mortality in the United States, and statin cholesterol-lowering
drugs have become the top-selling class of prescription drug.
Statin drugs generate more than $7 billion in annualized sales,
but these drugs have significant side effects. Vitamin C, with
annualize sales close to $180 million, has the very same
cholesterol-lowering property as the popular statin drugs.

Vitamin C Even Lowers Cholesterol - Naturally

In 1985, two years prior to the introduction of the popular statin
cholesterol-lowering drugs, the scientists who were investigating
the enzymes that help the body produce cholesterol, made an
important discovery: Vitamin C is a powerful anti-cholesterol
agent. The vitamin C molecule inhibits the same enzyme, HMG CoA
Reductase, that the statin cholesterol-lowering drugs inhibit. [*]

Individuals using home cholesterol-monitoring devices, such as the
LifeStream® monitor which is available in 55,000 retail outlets,
report that 6000 mg to 10,000 mg of vitamin C may be required for
maximum cholesterol lowering effect.

Statin Drugs May Cause Heart Failure Leading to Heart
Transplants

The structure of Coenzyme Q10 (CoQ10 or ubiquinone) was determined
by the Merck scientist Karl Folkers after its discovery in 1957.
[*] There have been at least 35 clinical studies showing CoQ10's
massive benefits for heart patients, especially patients in heart
failure. [*] And in Japan, until last year, CoQ10 was a heart
medication only available by prescription.

The drug giant, Merck, learned during their cholesterol-lowering
research that statin drugs block the body's production of its own
CoQ10. This blockage of CoQ10 synthesis is a serious action of
statins that causes fatigue, muscle pain and skeletal myopathy, (a
grave deterioration of muscle). Drug advertisements in Canada must
carry the CoQ10 statin-depletion warning, but the American FDA
does not require these important warnings, keeping U.S. medical
doctors in the dark and putting their patients at risk. [*]

Merck has more than one 1990 patent for adding CoQ10 to statins as
a means of circumventing the issue of blocking CoQ10 biosynthesis.
[United States Patent 4,933,165 ] Their having these patents since
1990 is proof that members of Merck corporation have been aware
that statins cause muscle deterioration. (The Merck Patents were
never implemented, probably because the world supply of CoQ10 is
far too limited to supply all statin drug users).

It is sad and truly frightening that today's hottest selling class
of prescription drugs, statin cholesterol-lowering drugs, are
known to deplete CoQ10 synthesis, yet these drugs are routinely
prescribed to heart patients!

Another Statin Side-Effect? Transient Global Amnesia

Former NASA astronaut Duane Graveline, M.D., believes that the
statin drug Lipitor® caused his own case of Transient Global
Amnesia (TGA) [http://www.spacedoc.net/Statins_flyer.html].
Graveline believes that these drugs are the cause of a recent
epidemic of TGA to hit emergency rooms, and fearing the dire
possibilities with airline pilots who take statins, Dr. Graveline
has begun a crusade to educate the medical profession and public
about the potential danger of cholesterol lowering drugs. The
vitamin C foundation has posted its collection of concerns at
www.vitamincfoundation.org/statinalert.

Chronic Scurvy Verified by CardioRetinometry

It has long been known that human arteries weaken without vitamin
C and other necessary nutritional support. Atheromas, or soft
atherosclerotic plaques, are the names given to abnormal
formations that appear in arteries. Dr. Pauling and associates
theorized with Willis that such plaque formations serve to
strengthen arteries, because they appear most often where the
blood pressure is highest. Sometimes a weak artery ruptures and
the resulting clot causes a heart attack or stroke. This condition
is most properly characterized as chronic scurvy.

Atheromas in the microscopic arteries in the retina have been
clearly visible to eye doctors, but until recently, they did not
believe that such build-ups were reversible. Dr. Sidney Bush,
DOpt, of the United Kingdom, accidentally discovered that
atheromas can be reversed in those patients instructed to take
from 3,000mg to 10,000mg of vitamin C. Dr. Bush made his discovery
while studying eye infections in contact lenses wearers. Vitamin C
was being tested as a preventive measure for these infections and,
serendipitously, Dr. Bush noticed that atheromas disappeared in
the study patients taking vitamin C.

Dr. Bush reports that some patients require as much as 10,000mg
daily to reverse soft atheromas.

Dr. Bush has invented a new diagnostic technique that he calls
CardioRetinometry. He believes this method of diagnosis will
revolutionize cardiology.

A new diagnostic technique can access coronary heart disease risk
(CHD) suggested by universal retinal arterial atheroma, previously
unsuspected as reversible. Physicians have overlooked, and
Optometrists/Opthamologists were not expecting that vitamin C
would have this effect. This effect was accidentally found and
linked to the vitamin C that contact lens wearers had agreed to
take. We have increasingly noticed it from 1999 using Retinometry
in the Hull Contact Lens and Eye Clinic. Such a discovery requires
urgent evaluation."

Dr. Bush has also promoted the idea that chronic scurvy not only
exists, but can be accurately measured. Eye doctors can now easily
diagnose this condition by examining the microscopic arteries
behind the eye before any symptoms of heart disease manifest.

Thanks to Dr. Bush we now know that vitamin C will reverse the
condition, in short order at the optimal dosage determined by
Cardioretinometry.

People today are under the extremely seriously mistaken impression
that nobody dies of scurvy any more! These studies may prove that
we are all dying faster from scurvy than hitherto suspected

The pericorneal vasculature, studied frequently by contact lens
practitioners, shows that Scurvy affects all humans some of the
time and most of us most of the time.

Cardioretinometry clearly demonstrates the relationship between
vitamin C intake and "atheromas" - plaques forming on the arteries
that serve the retina in the eye. Dr. Bush has published before
and after pictures taken with his new method and advocates the
need for rigorous studies.

The atheroma of the retinal arteries is a virtually perfect
surrogate outcome predictor of coronary heart disease and will
continue to be so as long as the eyes are connected to the rest of
the system. The modern electronic eye camera/microscopes with high
definition magnification facility show the impacting of the
cholesterol beautifully and also its redissolving into the
bloodstream when the system is restored to balance. And this is
seen in arterioles too small to be seen with the naked eye!

Whilst day to day variations in the pericorneal vessels are a
relatively easily readable ‘barometer’ of ‘ephemeral’ scurvy
especially when viewed via the slit lamp biomicroscope of the
contact lens practitioner, little attention has been paid to it
except by a few dedicated medical practitioners.

The pericorneal arterioles and capillaries can and are graded in
my system of practice into ten degrees of scurvy allowing the
accurately prediction to patients of how much or little vitamin C
they have been eating. The highest mark anybody has had is 94%
When I started this grading, c.1997, I confounded my nursing staff
by being able to correctly identify patients who ate no or few
greens. But the same ease of observation does not attach to
identification of the chronic subclinical variety. It cannot
identify dietary faults in the most recent past. In a similar way
to slow build-up of vitamin E in the body fat and cell walls of
the brain, it takes over a month to be sure what is happening to
the cholesterol in the retinal arteries.

Dr. Bush now has evidence that even calcified "hard" plaques can
be reversed over the course of 2-years on a high vitamin C intake.
This development throws a hammer into the Government/CODEX
'recommended' daily allowance of 60 mg and the 2000mg maximum
tolerable allowance.

The moral of the story is to have regular examinations of the
retinal arteries by a patient, suitably equipped optometrist
trained in CardioRetinometry. This is in my opinion, after five
years of observation of my patients' health, the most valuable
safeguard of one's cardiovascular and probably many other systems
as they do not act in isolation.

Our Path to the World of Vitamin C and Other Discoveries

In 1994, no one knew whether Linus Pauling's high vitamin C/lysine
protocol really worked. Our company, Intelisoft Multimedia, Inc.,
had obtained the rights to the Pauling video on heart disease [*]
and tried to promote it. At that time, the author had no financial
interest in any nutritional product. Years later, we realized that
Pauling's close associate, Matthias Rath, apparently had not fully
appreciated what Pauling had been advocating. For this reason we
took on the task of promulgating Pauling's discoveries. Tower
Laboratories Corporation was willing to promote a new product with
sufficiently high doses of vitamin C and lysine, which matched
Pauling's dosage recommendations.

From the beginning, the Pauling therapy began to absolutely cure
the incurable - miracle after miracle. Many of these testimonials
have been written and are posted at the video web site
PaulingTherapy.com. Yet, none of the old media printed a word
about this phenomenon.

Kevin Trudeau has written a new book entitled Natural Cures 'They'
Don't Want You to Know About ( naturalcures.com). Most of the
information in this book dovetails with our experience advocating
the Pauling therapy for heart disease during the past decade.
Trudeau points out that it is against Federal law to tell people
that a particular product cures a disease without consent from the
Government. Kevin is mad, and I feel the same way.

Almost everyone in authority concerned with heart disease knows,
or has been advised, except patients, that Pauling's theory is
undeniable and that his recommended therapy works quickly.

The safe and effective answer to the most common form of heart
disease - plaques forming over weak arteries - is 6000mg to
18000mg vitamin C to strengthen the arteries. And Dr. Pauling
invention of administering high-dose lysine resolves existing
plaques. This combination appears to work in most individuals
within 10 days, with the correct dosage

The general attitude of traditional medicine is wrong and
self-serving on these matters. Instead of depriving patients of a
potentially life-saving therapy until the 'necessary' (and, as yet
still unplanned) studies are run, doctors should be recommending
Dr. Pauling's therapy to all heart patients until there is
evidence this non- toxic therapy doesn't work. We have heard
reports of cardiologists becoming livid after having been briefed
on Pauling's theories by respected sources. These doctors
expressed verbal frustration at their medical journals for not
informing them of these developments.

The Solution to Other Forms of Heart DiseaseHeart FailureMany people experience a remission from heart failure after
they adopt Pauling's vitamin C and lysine therapy. However, there
is much evidence that the cause of heart failure in most people is
a Coenzyme Q10 deficiency. This vitamin-like coenzyme is required
in our fuel-cells, the mitochondria, in order to manufacture the
body's fuel Adenine Triphosphate (ATP).

Several other vitamins are required for the human body to produce
its own CoQ10, and humans are known to synthesize less CoQ10 as we
age. Scores of prescription drugs, and in fact all the statin
cholesterol-lowering drugs, block the body's production of CoQ10!
Therefore, it can be accurately stated that these drugs, given to
most heart patients, cause a form of heart disease - heart
failure. The rate of heart failure has tripled, and CoQ10 experts
cite studies which attribute this increase to higher dosages of
statins. [*]

Normally, blood pressure elevates during times of stress
(fight-or-flight) for short periods. The higher blood pressure
ensures that glucose and other nutrients enter the cells in order
to aid response to the stress. It is also normal for high blood
pressure to normalize after the stressful event passes. Generally,
doctors measure blood pressure because a small narrowing of the
artery has an exponential effect on hypertension. This blood
pressure reading is considered an indicator of (weak) arterial
plaque. According to discussions in the British Medical Journal,
opthamologists have noticed that the plaques form in microscopic
retinal arteries before the onset of elevated blood pressure.

Pauling's therapy has been an effective treatment for
hypertension, as are other nutrients, such as magnesium, vitamin
B6, the amino acid arginine and several other orthomolecular
approaches. Health journalist Bill believes that 200 mg of vitamin
B6 is more effective than many prescription drugs for
hypertension. [http://www.askbillsardi.com/sdm.asp?pg=hyper_1 ]

Calcified Arteries

Many heart patients have hard or calcified arteries. This
condition makes heart attack more likely because blood vessels are
unable to dilate properly in the event of a clot or blockage. The
most probable cause of excess calcium building up-in the arteries
of heart patients is the use of prescription blood thinners. These
prescription medications either simulate or block vitamin K and
they are routinely prescribed. There are over 200 MEDLINE studies
as evidence of our suspicion that this is a fact. [
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Display&dopt=pubmed_pubmed&from_uid=9743228
] High dose vitamin K reduces calcium in soft tissues and is
considered a standard treatment for osteoporosis in Japan. The
vitamin acts as a hormone and helps move calcium from soft tissues
into bone. [http://www.lef.org/magazine/mag2000/feb00-report.html]

Chelation doctors deserve great respect. Their EDTA detoxification
therapy benefits many patients, but EDTA will not cure heart
disease by itself. The Chelation treatments that are effective
incorporate a supplement program which includes vitamin C.

Heart Attack

Strong vitamin C/lysine-fortified arteries are less likely to
rupture. If there is no rupture, there will be no clot. If there
is no clot, there will be no heart attack caused by a blockage of
blood to the heart.

World Health Organization (WHO) researchers have discovered that
low serum vitamin E is a 70% better predictor of heart attack than
either hypertension (high blood pressure) or high cholesterol.
Also, Teo and other researchers have discovered that a magnesium
injection immediately after a heart attack saved 55% of those who
would have died. (Placebo controlled trial)
[http://www.internetwks.com/pauling/jon.html ].

Congenital Heart Defects and Heart Damage

We have documented extraordinary cases of patients whose damage
hearts, as measured by EKG, have returned to normal. Harvard
medical researchers found that vitamin C was the only one of 880
substances tested that caused heart muscle cells to regenerate
from stem cells. [
http://www.sciencedaily.com/releases/2003/04/030401073122.htm]

It is our experience that a good natural vitamin E with mixed
tocopherals and tocotrienols (such as 2000 IU Unique-E from A. C.
Grace), in conjunction with high vitamin C, as ascorbic acid, is
required for EKG reversals. Such a reversal occurred in 3 months.
[http://www.internetwks.com/carolsmith]

Cardiology: A Real Nightmare Scenario

Heart patients have the right to be highly skeptical or even
fearful of their cardiologist. With the exception of the
nitroglycerine patch [*], there are no standard heart medications
or treatments that do help heart patients. In our opinion, all
prescription treatments, of which we are aware, make patients
worse by blocking CoQ10, or by causing rapid calcification of soft
tissues, by making blood clots more likely, or by raising